CN105102003B - Tall and erect-anti-PSMA antibody conjugates of Pyrrolobenzodiazepines - Google Patents

Tall and erect-anti-PSMA antibody conjugates of Pyrrolobenzodiazepines Download PDF

Info

Publication number
CN105102003B
CN105102003B CN201380065405.7A CN201380065405A CN105102003B CN 105102003 B CN105102003 B CN 105102003B CN 201380065405 A CN201380065405 A CN 201380065405A CN 105102003 B CN105102003 B CN 105102003B
Authority
CN
China
Prior art keywords
seq
group
antibody
pairs
acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN201380065405.7A
Other languages
Chinese (zh)
Other versions
CN105102003A (en
Inventor
贝克尔 帕特里修斯·亨德里克斯·科内利斯·范
菲利普·威尔逊·霍华德
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ADC Therapeutics SA
Original Assignee
ADC Therapeutics SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ADC Therapeutics SA filed Critical ADC Therapeutics SA
Publication of CN105102003A publication Critical patent/CN105102003A/en
Application granted granted Critical
Publication of CN105102003B publication Critical patent/CN105102003B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Abstract

It is bound to the antibody of PSMA and the conjugate of PBD dimer.

Description

Tall and erect-anti-PSMA antibody conjugates of Pyrrolobenzodiazepines
Technical field
The present invention relates to have with the pyrrolo- benzo of the unstable C2 or N10 blocking group of the joint form with antibody Diaza(Pyrrolobenzodiazepines are tall and erect, pyrrolobenzodiazepines) (PBD).
Background technique
Pyrrolobenzodiazepines
Some Pyrrolobenzodiazepines(PBD) it can identify and be incorporated into the particular sequence of DNA；Preferably sequence is PuGPu.The first PBD antitumor antibiotics, Anthramycin (anthramycin) (Leimgruber, et are found in nineteen sixty-five al.,J.Am.Chem.Soc.,87,5793-5795(1965)；Leimgruber,et al.,J.Am.Chem.Soc.,87, 5791-5793(1965)).Thereafter, it has reported the PBD of many naturally occurrings, and has developed for various analogs more than 10 Kind route of synthesis (Thurston, et al., Chem.Rev.1994,433-465 (1994)；Antonow,D.and Thurston,D.E.,Chem.Rev.2011111(4),2815-2864).Family member includes abbeymycin (Hochlowski, et al., J.Antibiotics, 40,145-148 (1987)), odd Ka-7038Ⅶ (chicamycin) (Konishi, et al., J.Antibiotics, 37,200-206 (1984)), DC-81 (Japan Patent 58-180487； Thurston,et al.,Chem.Brit.,26,767-772(1990)；Bose,et al.,Tetrahedron,48,751- 758 (1992)), mazethramycin (mazethramycin) (Kuminoto, et al., J.Antibiotics, 33,665- 667 (1980)), neothramycin (neothramycin) A and B (Takeuchi, et al., J.Antibiotics, 29,93- 96(1976))、porothramycin(Tsunakawa,et al.,J.Antibiotics,41,1366-1373(1988))、 prothracarcin(Shimizu,et al,J.Antibiotics,29,2492-2503(1982)；Langley and Thurston, J.Org.Chem., 52,91-97 (1987)), sibanomicin (sibanomicin) (DC-102) (Hara, et al.,J.Antibiotics,41,702-704(1988)；Itoh,et al.,J.Antibiotics,41,1281-1284 (1988)), sibiromycin (sibiromycin) (Leber, et al., J.Am.Chem.Soc., 110,2992-2993 And tomamycin (tomamycin) (Arima, et al., J.Antibiotics, 25,437-444 (1972)) (1988)).PBD For following general structure:
Their difference is the quantity, type and position of substituent group, is their aromatics A ring and pyrrolo- C ring two Person, and it is C ring filling degree.In B ring, at the position N10-C11 (it is responsible for making the alkylated electrophilic center of DNA) There are imines (N=C), carbinolamine (carbinolamine) (NH-CH (OH)) or carbinolamine methyl ethers (NH-CH (OMe)).It is all Known natural products has (S)-configuration at the chiral position C11a, and when from from C ring to A ring, which mentions to them (right-handed twist) is reversed for the right hand.This give they for the ditch of B-form DNA etc. helicities (isohelicity) 3D shape appropriate obtains (Kohn, the In Antibiotics that is slidably matched at binding site III.Springer-Verlag,New York,pp.3-11(1975)；Hurley and Needham-VanDevanter, Acc.Chem.Res.,19,230-237(1986)).The ability that they form adduct in ditch interferes them DNA processing, therefore they can be used as antitumor agent.
By Gregson et al. as compound 1 (Chem.Commun.1999,797-798) and by Gregson et Al. particularly advantageous Pyrrolobenzodiazepines are described as compound 4a (J.Med.Chem.2001,44,1161-1174)Compound.The compound, also referred to as SG2000, described below:
WO 2007/085930 is described with the dimerization for being connected to the linker group of cell binding agent (such as antibody) The preparation of body PBD compound.Connector is present in the bridge of the monomer PBD unit of connection dimer.
The present inventor has been described in WO 2011/130613 and WO 2011/130616 to be had for being connected to carefully The dimer PBD compound of the linker group of born of the same parents' bonding agent (such as antibody).Connector in these compounds is connected to by C2 PBD core, and usually cut by the effect of enzyme in linker group.In WO 2011/130598, connecing in these compounds Head is connected to one in N10 obtainable on PBD core, and is usually cut by the effect of enzyme in linker group.
Antibody-drug conjugates
It has built up for the antibody therapy with cancer, immunological diseases and the targeted therapy of vascular disease (Carter,P.(2006)Nature Reviews Immunology 6:343-357).It is thin for local delivery in treatment of cancer Cellular toxicity agent or cytostatics (that is, drug) are to kill or inhibit the antibody-drug conjugates (ADC) of tumour cell (that is, exempting from Epidemic disease conjugate) use target drug moiety be delivered to tumour, and intracellular accumulation wherein, and these non-binding drugs The whole body of reagent, which is given, can cause to the unacceptable toxic level of normal cell (Xie et al (2006) Expert.Opin.Biol.Ther.6(3):281-291；Kovtun et al(2006)Cancer Res.66(6):3214- 3121；Law et al(2006)Cancer Res.66(4):2328-2337；Wu et al(2005)Nature Biotech.23(9):1137-1145；Lambert J.(2005)Current Opin.in Pharmacol.5:543-549； Hamann P.(2005)Expert Opin.Ther.Patents 15(9):1087-1103；Payne,G.(2003)Cancer Cell 3:207-212；Trail et al(2003)Cancer Immunol.Immunother.52:328-337；Syrigos and Epenetos(1999)Anticancer Research 19:605-614)。
It hereby is observed that the greatest treatment efficacy with minimum toxicity.Design and the effort for improving ADC concentrate on monoclonal antibody (mAb) in selectivity and mechanism of drug action, drug connection, drug/Antibody ratio (load) and drug release property (Junutula,et al.,2008b Nature Biotech.,26(8):925-932；Dornan et al(2009)Blood 114(13):2721-2729；US 7521541；US 7723485；WO2009/052249；McDonagh(2006)Protein Eng.Design&Sel.19(7):299-307；Doronina et al(2006)Bioconj.Chem.17:114-124； Erickson et al(2006)Cancer Res.66(8):1-8；Sanderson et al(2005)Clin.Cancer Res.11:843-852；Jeffrey et al(2005)J.Med.Chem.48:1344-1358；Hamblett et al (2004)Clin.Cancer Res.10:7063-7070).Drug moiety can be by including that micro-pipe combines, DNA is combined, albumen The mechanism that enzyme body and/or topoisomerase inhibit assigns their cytotoxicities and cyto-inhibition.Be bound to big antibody or When protein receptor ligand, some cytotoxic drugs are intended to torpescence or compared with torpescence.
Present inventors have developed specific PBD homodimeric antibody conjugates.
Summary of the invention
The first aspect of the present invention includes formula L- (DL)pConjugate, wherein DLFor Formulas I or Formula II:
Wherein:
L is antibody as defined below (Ab)；
As C2 ' and C3 ' between there are when double bond, R12Selected from the group being made up of:
(ia)C5-10Aryl group, the one or more substituent groups being optionally chosen in self-contained below group replace: halogen Element, nitro, cyano, ether, carboxyl, ester, C1-7Alkyl, C3-7Heterocycle and double-oxygroup-C1-3Alkylidene；
(ib)C1-5Representative examples of saturated aliphatic alkyl；
(ic)C3-6Saturated cyclic alkyls；
(id)Wherein, R21、R22And R23Each of independently selected from H, C1-3Saturated alkyl, C2-3Alkene Base, C2-3Alkynyl and cyclopropyl, wherein R12The sum of carbon atom is no more than 5 in group；
(ie)Wherein, R25aAnd R25bIn one be H and another be selected from: phenyl, the phenyl are optional Ground is replaced by the group selected from halogen, methyl, methoxyl group；Pyridyl group；And thiophenyl；With
(if)Wherein, R24It is selected from: H；C1-3Saturated alkyl；C2-3Alkenyl；C2-3Alkynyl；Cyclopropyl；Phenyl, The phenyl is optionally replaced by the group selected from halogen, methyl, methoxyl group；Pyridyl group；And thiophenyl；
As C2 ' and C3 ' between there are when singly-bound,
R12It isWherein, R26aAnd R26bIndependently selected from H, F, C1-4Saturated alkyl, C2-3Alkenyl, the alkyl and Alkenyl group is optionally selected from C1-4Alkyl amino and C1-4The group of Arrcostab replaces；Alternatively, working as R26aAnd R26bIn one When being H, another is selected from nitrile and C1-4Arrcostab；
R6And R9Independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR ', nitro, Me3Sn and halogen；
Wherein, R and R ' is independently selected from optionally substituted C1-12Alkyl, C3-20Heterocycle and C5-20Aryl group；
R7Selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR ', nitro, Me3Sn and halogen；
R " is C3-12Alkylidene group, the chain can be interrupted by item below one or more: hetero atom, such as O, S, NRN2(wherein RN2It is H or C1-4Alkyl) and/or aromatic ring, such as benzene or pyridine；
Y and Y ' is selected from O, S or NH；
R6’、R7’、R9’It is respectively selected from and R6、R7And R9Identical group；
[Formulas I]
RL1’It is the connector for being connected to antibody (Ab)；
R11aSelected from OH, ORA, wherein RAIt is C1-4Alkyl and SOzM, wherein z is that 2 or 3 and M is pharmaceutically may be used for unit price The cation of receiving；
R20And R21Or it is formed together the double bond between the nitrogen and carbon atom that they are connected；Or
R20Selected from H and RC, wherein RCIt is end-capping group；
R21Selected from OH, ORAAnd SOzM；
When between C2 and C3 there are when double bond, R2Selected from the group being made up of:
(ia)C5-10Aryl group, the one or more substituent groups being optionally chosen in self-contained below group replace: halogen Element, nitro, cyano, ether, carboxyl, ester, C1-7Alkyl, C3-7Heterocycle and double-oxygroup-C1-3Alkylidene；
(ib)C1-5Representative examples of saturated aliphatic alkyl；
(ic)C3-6Saturated cyclic alkyls；
(id)Wherein, R11、R12And R13Each of independently selected from H, C1-3Saturated alkyl, C2-3Alkene Base, C2-3Alkynyl and cyclopropyl, wherein R2The sum of carbon atom is not more than 5 in group；
(ie)Wherein, R15aAnd R15bIn one be H, and another is selected from: phenyl, the benzene Base is optionally replaced by the group selected from halogen, methyl, methoxyl group；Pyridyl group；And thiophenyl；With
(if)Wherein, R14It is selected from: H；C1-3Saturated alkyl；C2-3Alkenyl；C2-3Alkynyl；Cyclopropyl；Benzene Base, the phenyl are optionally replaced by the group selected from halogen, methyl, methoxyl group；Pyridyl group；And thiophenyl；
When between C2 and C3 there are when singly-bound,
R2It isWherein, R16aAnd R16bIndependently selected from H, F, C1-4Saturated alkyl, C2-3Alkenyl, the alkyl Optionally C is selected from alkenyl group1-4Alkyl amino and C1-4The group of Arrcostab replaces；Or work as R16aAnd R16bIn one be When H, another is selected from nitrile and C1-4Arrcostab；
[Formula II]
R22It is formula III a, formula III b or formula III c:
(a)
Wherein, A is C5-7Aryl group, and
(i)Q1It is singly-bound, and Q2Selected from singly-bound and-Z- (CH2)n, wherein Z is selected from singly-bound, O, S and NH, and n is 1 To 3；Or
(ii)Q1It is-CH=CH-, and Q2It is singly-bound；
(b)
Wherein,
RC1、RC2And RC3Independently selected from H and unsubstituted C1-2Alkyl；
(C)
Wherein, Q is selected from O-RL2’、S-RL2’And NRN-RL2’, and RNSelected from H, methyl and ethyl；
It includes below group: O-R that X, which is selected from,L2’、S-RL2’、CO2-RL2’、CO-RL2’, NH-C (=O)-RL2’、NHNH-RL2’、 CONHNH-RL2’、 NRNRL2’, wherein RNSelected from including H and C1-4Alkyl Group；
RL2’It is the connector for being connected to antibody (Ab)；
R10And R11Or it is formed together the double bond between the nitrogen and carbon atom that they are connected；Or
R10It is H and R11Selected from OH, ORAAnd SOzM；
R30And R31Or it is formed together the double bond between the nitrogen and carbon atom that they are connected；Or
R30It is H and R31Selected from OH, ORAAnd SOzM。
In some embodiments, conjugate is not:
ConjA
ConjB
ConjC:
ConjD
ConjE:
In other embodiments, it may be preferred to conjugate be selected from formula ConjA, ConjB, ConjC, ConjD and The conjugate of ConjE.
Subscript p in Formulas I is integer of 1 to 20.Therefore, conjugate includes to be covalently attached at least one by connector unit The antibody (Ab) as defined below of a drug unit.It is the targeting agent for being bound to targeting moiety with body unit, hereafter carries out A more complete description.Therefore, the present invention also provides the methods for treating for example various cancers and autoimmune disease.Drugloading rate (drug loading) is indicated by p, is the number of every antibody drug molecule.Drugloading rate can be in 1 to 20 drug unit (DLIn the range of)/antibody.For composition, p indicates the drug loading of conjugate in the composition, and p is 1 to 20 In range.
The second aspect of the present invention provides the method for preparation conjugate according to the first aspect of the invention, including by Formulas IL Or Formula IILCompound:
It is bound to antibody as defined below (Ab), in which:
RL1It is the connector for being suitable for connection to antibody (Ab)；
R22LIt is formula III aL, formula III bLOr formula III cL:
(a)
(b)
(c)
Wherein, QLSelected from O-RL2、S-RL2And NRN-RL2, and RNSelected from H, methyl and ethyl；
XLSelected from including below group: O-RL2、S-RL2、CO2-RL2、CO-RL2, N=C=O-RL2、NHNH-RL2、CONHNH- RL2、NRNRL, wherein RNSelected from including H and C1-4The group of alkyl；
RL2It is the connector for being suitable for connection to antibody (Ab)；
And what all residue group such as first aspects limited.
Therefore, in second aspect it may be preferred that the present invention provide preparation selected from by ConjA, ConjB, ConjC, The method of the conjugate of the group of ConjD and ConjE composition, including A will be respectively selected from:
B:
C:
D:
And E:
Compound in conjunction with antibody as defined below.
WO 2011/130615 discloses compound 26:
It is the parent compound of A.Compound A includes the PBD and the connector for being connected to cell binding agent.Cell knot Mixture provides multiple ethylene glycol moieties to provide dissolubility, is useful in the synthesis of conjugate.
WO 2010/043380 and WO 2011/130613 disclose compound 30:
WO 2011/130613 also discloses compound 51:
Compound B and 30 difference of compound are: only having (CH between the part PBD2)3Chain (tether), rather than (CH2)5Chain, this reduces the lipophilicity of the PBD dimer of release.Linking group is in contraposition rather than meta position is connected to C2- phenyl base Group.
WO 2011/130613 discloses compound 93:
Compound C is different from both ways.Cell binding agent provides the ethylene glycol moieties for increasing number to provide dissolution Property, this is useful in the synthesis of conjugate, and phenyl substituent provides two rather than an oxygen atom, this is also contributed to Dissolution.The structure of compound C can also mean that it is more strongly incorporated in ditch.
Compound A, B and C have on each C ring there are two sp2Center can permit than only having in each C ring One sp2Stronger combination of the compound at center in DNA ditch.
WO 2011/130598 discloses compound 80:
Compound D is different from by the inclusion of the iodo-acetamide for being connected to cell binding agent.The group is being tied In terms of stability when being bonded to cell binding agent, the advantage (see below) more than compound 80 can be provided.In compound 80 Maleimide base group reverse Michael can be undergone to react, become not in conjunction with cell binding agent, thus be easy to by comprising The other biological molecule (such as albumin and glutathione) of mercaptan is removed.It cannot occur this not combine with compound A.In addition, Iodoacetamido amine groups can avoid other undesirable side reactions.
Compound E by below with disclosed PBD dimer (the drug connector for having C2-3 cyclic olefinic bond) before no It is same: to there is lesser, more alipotropic C2 substituent group, for example, 4F- phenyl, propylidene.In this way, conjugate (the ginseng of compound B See following) less tend to assemble in synthesis.This poly- of conjugate can be measured by size exclusion chromatography (SEC) Collection.
Compound D and E has in each C ring there are two sp2Center, this can permit than only having in each C ring One sp2Stronger combination of the compound at center in DNA ditch.
It can will be in WO 2010/043880, WO 2011/130613, WO 2011/130598 and WO 2011/130616 Disclosed drug connector is used for this invention and incorporated herein by reference.It can the conjunction according to described in these disclosures At drug connector described herein.
Specific embodiment
The present invention in terms of PBD compound to be provided to subject to preferred position suitable for using.Conjugate allows Release does not retain any portion of activity PBD compound of connector.There is no the short of the reactivity that can influence PBD compound Cut (residual, stub).Therefore, ConjA will discharge compound R elA:
ConjB will discharge compound R elB:
ConjC will discharge compound R elC:
ConjD will discharge compound R elD:
And ConjE will discharge compound R elE:
PBD dimer in the present invention and the given joint between antibody are preferably extracellularly stable.Conveying or It is deliverrf into before cell, antibody-drug conjugates (ADC) are preferably stable and keep complete, that is, antibody keeps connecting It is connected to drug moiety.Connector is stable outside target cell, and can be cut in the cell with effective speed.Effective connector Will: the specific binding characteristics of (i) holding antibody；(ii) allow the Intracellular delivery of conjugate or drug；(iii) it keeps stablizing With it is complete, that is, until conjugate be delivered or be delivered to it target site just cut；And (iv) keeps PBD drug portion Cytotoxic effect, killing functions of immunocytes or the cyto-inhibition divided.Can by standard analytical techniques such as mass spectrum, HPLC and Separation/analytical technology LC/MS measurement ADC stability.
Through enzyme (such as cathepsin) on linking group and especially on val-ala dipeptide moieties Effect, formula ConjA, ConjB, ConjC, ConjD or ConjE conjugate expectation activation site at realize formula The delivering of the compound of RelA, RelB, RelC, RelD or RelE.
Antibody
On the one hand, antibody is the antibody for being bound to PSMA, which includes to have according to SEQ ID NO.1,3,5,7,8,9 Or in 10 any sequence VH structural domain.
Antibody can further include VL structural domain.In some embodiments, antibody, which further includes, has according to SEQ The VL structural domain of any sequence in ID NO.2,4,6,11,12,13,14,15,16,17 or 18.
In some embodiments, antibody includes the VH structural domain pairs of with VL structural domain, VH structural domain and VL structural domain With the sequence selected from the group being made up of:
With pairs of SEQ ID NO.1 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.3 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.5 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.7 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.8 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.9 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18； Or
With pairs of SEQ ID NO.10 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18. For example, SEQ ID NO.1 pairs of with SEQ ID NO.2 and SEQ ID NO.4 pairs of SEQ ID NO.3 and SEQ ID NO.6 pairs of SEQ ID NO.5 and SEQ ID NO.11 pairs of SEQ ID NO.7 and SEQ ID NO.12 pairs of SEQ ID NO.7 and SEQ ID NO.13 pairs of SEQ ID NO.7 and SEQ ID NO.14 pairs of SEQ ID NO.7 and SEQ It is ID NO.15 pairs of SEQ ID NO.7, SEQ ID NO.7 pairs of with SEQ ID NO.16, pairs of with SEQ ID NO.17 SEQ ID NO.7, with SEQ ID NO.18 pairs of SEQ ID NO.7, with SEQ ID NO.11 pairs of SEQ ID NO.8 and SEQ ID NO.12 pairs of SEQ ID NO.8 and SEQ ID NO.13 pairs of SEQ ID NO.8 and SEQ ID NO.14 pairs of SEQ ID NO.8, with SEQ ID NO.15 pairs of SEQ ID NO.8, it is pairs of with SEQ ID NO.16 SEQ ID NO.8 and SEQ ID NO.17 pairs of SEQ ID NO.8 and SEQ ID NO.18 pairs of SEQ ID NO.8, Pairs of SEQ ID NO.9 and SEQ ID NO.12 pairs of SEQ ID NO.9 and SEQ ID with SEQ ID NO.11 NO.13 pairs of SEQ ID NO.9, with SEQ ID NO.14 pairs of SEQ ID NO.9, it is pairs of with SEQ ID NO.15 SEQ ID NO.9 and SEQ ID NO.16 pairs of SEQ ID NO.9 and SEQ ID NO.17 pairs of SEQ ID NO.9, Pairs of SEQ ID NO.9 and SEQ ID NO.11 pairs of SEQ ID NO.10 and SEQ ID with SEQ ID NO.18 NO.12 pairs of SEQ ID NO.10, with SEQ ID NO.13 pairs of SEQ ID NO.10, it is pairs of with SEQ ID NO.14 SEQ ID NO.10 and SEQ ID NO.15 pairs of SEQ ID NO.10 and SEQ ID NO.16 pairs of SEQ ID The NO.10 and SEQ ID NO.17 pairs of SEQ ID NO.10 or SEQ ID NO.10 pairs of with SEQ ID NO.18.
One or more VH structural domains and VL structural domain can be in pairs to form the antibody antigen-binding site for combining PSMA.
In some embodiments, antibody is the complete antibody comprising the VH structural domain pairs of with VL structural domain, VH structure Domain and VL structural domain have the sequence selected from the group being made up of:
With pairs of SEQ ID NO.1 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.3 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.5 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.7 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.8 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18；
With pairs of SEQ ID NO.9 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18； Or
With pairs of SEQ ID NO.10 any in SEQ ID NO.2,4,6,11,12,13,14,15,16,17 or 18. For example, SEQ ID NO.1 pairs of with SEQ ID NO.2 and SEQ ID NO.4 pairs of SEQ ID NO.3 and SEQ ID NO.6 pairs of SEQ ID NO.5 and SEQ ID NO.11 pairs of SEQ ID NO.7 and SEQ ID NO.12 pairs of SEQ ID NO.7 and SEQ ID NO.13 pairs of SEQ ID NO.7 and SEQ ID NO.14 pairs of SEQ ID NO.7 and SEQ It is ID NO.15 pairs of SEQ ID NO.7, SEQ ID NO.7 pairs of with SEQ ID NO.16, pairs of with SEQ ID NO.17 SEQ ID NO.7, with SEQ ID NO.18 pairs of SEQ ID NO.7, with SEQ ID NO.11 pairs of SEQ ID NO.8 and SEQ ID NO.12 pairs of SEQ ID NO.8 and SEQ ID NO.13 pairs of SEQ ID NO.8 and SEQ ID NO.14 pairs of SEQ ID NO.8, with SEQ ID NO.15 pairs of SEQ ID NO.8, it is pairs of with SEQ ID NO.16 SEQ ID NO.8 and SEQ ID NO.17 pairs of SEQ ID NO.8 and SEQ ID NO.18 pairs of SEQ ID NO.8, Pairs of SEQ ID NO.9 and SEQ ID NO.12 pairs of SEQ ID NO.9 and SEQ ID with SEQ ID NO.11 NO.13 pairs of SEQ ID NO.9, with SEQ ID NO.14 pairs of SEQ ID NO.9, it is pairs of with SEQ ID NO.15 SEQ ID NO.9 and SEQ ID NO.16 pairs of SEQ ID NO.9 and SEQ ID NO.17 pairs of SEQ ID NO.9, Pairs of SEQ ID NO.9 and SEQ ID NO.11 pairs of SEQ ID NO.10 and SEQ ID with SEQ ID NO.18 NO.12 pairs of SEQ ID NO.10, with SEQ ID NO.13 pairs of SEQ ID NO.10, it is pairs of with SEQ ID NO.14 SEQ ID NO.10 and SEQ ID NO.15 pairs of SEQ ID NO.10 and SEQ ID NO.16 pairs of SEQ ID The NO.10 and SEQ ID NO.17 pairs of SEQ ID NO.10 or SEQ ID NO.10 pairs of with SEQ ID NO.18.
In some embodiments, the antibody with for be bound to PSMA by hybridoma ATCC accession number HB-12126 point The antibody competition secreted.On the other hand, the antibody with for be bound to PSMA by hybridoma ATCC accession number HB-12109 secrete Antibody competition.In one embodiment, antibody with 2,5 times or 10 times not less than the antibody secreted by hybridoma compared with Low association constant (Ka) combine PSMA.
On the one hand, antibody is the antibody secreted by hybridoma.In one embodiment, hybridoma is ATCC accession number B- 12126.In another embodiment, hybridoma is ATCC accession number HB-12109.
In some embodiments, antibody is J415 antibody disclosed in WO02/098897.
In some embodiments, antibody is J519 antibody disclosed in WO02/098897.
On the one hand, antibody is antibody described herein, is modified (or further modification) as described below.? In some embodiments, antibody is the humanization of antibody disclosed herein, deimmunizes (deimmunised) or resurfacing (resurfaced) variant.For example, (i) comprising the VH structural domain SEQ ID NO.1 pairs of with VL structural domain SEQ ID NO.2 Antibody, J519 antibody disclosed in (ii) WO02/098897, (iii) are secreted anti-by hybridoma ATCC accession number HB-12109 Body, (iv) include the antibody of the VH structural domain SEQ ID NO.5 pairs of with VL structural domain SEQ ID NO.6, (v) WO02/ J415 antibody disclosed in 098897, (vi) by the hybridoma accession number HB-12126 antibody secreted humanization, go it is immune or The variant of resurfacing.
Term
The term " antibody " of this paper be use and specifically cover in the broadest sense monoclonal antibody, polyclonal antibody, Dimer, polymer, multi-specificity antibody (for example, bispecific antibody), complete antibody and antibody fragment, as long as they are showed Desired bioactivity out, for example, in conjunction with ability (Miller et al (2003) Jour.of Immunology of PSMA 170:4854-4861).Antibody can be murine antibodies, human antibodies, humanized antibody, chimeric antibody or come from other The antibody of species.Antibody is the protein generated by immune system that can identify and be bound to specific antigen.(Janeway, C.,Travers,P.,Walport,M.,Shlomchik(2001)Immuno Biology,5th Ed.,Garland Publishing,New York).Target antigen usually has many binding sites, also referred to as epitope, by Multiple Antibodies CDR is identified.Every kind of antibody for being specifically bound to different epitopes has different structures.Therefore, a kind of antigen can have More than one correspondence antibody.Antibody includes the immunocompetence portion of full-length immunoglobulin molecule or full-length immunoglobulin molecule Point, that is, the molecule of the antigen binding site of antigen of interested target or part thereof, this target packet are combined comprising immunologic specificity It includes but is not limited to cancer cell or generate the cell of autoimmune antibody relevant to autoimmunity disease.Immunoglobulin can be Any form (for example, IgG, IgE, IgM, IgD and IgA), classification (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) Or subclass or allograft (allotype) are (for example, mankind G1m1, G1m2, G1m3, non-G1m1 are [that is, in addition to G1m1 Any isoantigen immunoglobulin], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, Km1, Km2 and Km3) immunoglobulin molecules.Exempt from Epidemic disease globulin can be originated from any species, including source of people, source of mouse or rabbit source.
As it is used herein, " in conjunction with PSMA " is higher than non-specific companion's such as bovine serum albumin(BSA) for referring to antibody (BSA, Genbank accession number CAA76847, version number CAA76847.1GI:3336842 record update date: January 7 in 2011 Day, 02:30PM) affinity combination PSMA.In some embodiments, when measuring under the conditions of physiological solution, antibody is with height In the antibody association constant at least 2 for BSA, 3,4,5,10,20,50,100,200,500,1000,2000,5000,104、105 Or 106Association constant (K againa) combine PSMA.Antibody of the invention can be with high-affinity combination PSMA.For example, in some realities It applies in mode, antibody can be to be equal to or less than about 10-6M, such as 1 × 10-6、10-7、10-8、10-9,10-10、10-11、10-12、10 -13Or 10-14KDIn conjunction with PSMA.
As it is used herein, PSMA refers to prostate-specific membrane antigen.In one embodiment, PSMA polypeptide pair Update date: June 23 in 2010 should be recorded in Genbank accession number AAA60209, version number AAA60209.1GI:190664 Day, 08:48AM.In one embodiment, the nucleic acid for encoding PSMA polypeptide corresponds to Genbank accession number M99487, version Number M99487.1GI:190663 records update date: on June 23rd, 2010,08:48AM.
" antibody fragment " includes a part of full length antibody, usually its antigen binding domain or variable region.Antibody fragment Example includes Fab, Fab', F (ab')2With scFv segment；Binary；Linear antibodies；The segment generated by Fab expression library resists only Special type (anti-Id) antibody, CDR (complementary determining region) and any above immunologic specificity are bound to cancer cell antigen, viral antigen Or the epitope binding fragments of microbial antigen, single-chain antibody molecules, and the multi-specificity antibody formed by antibody fragment.
As used in this article, term " monoclonal antibody " refers to obtained from the anti-of the substantially group of homogeneity antibody Body, that is, in addition to the possible naturally occurring mutation that can be existed in a small amount, the single antibody including the group is identical 's.Monoclonal antibody is high degree of specificity for single antigen site.In addition, relative to polyclonal antibody preparations comprising For the different antibodies of different determinants (epitope), each monoclonal antibody is for the single determinant on antigen.Except they Other than specificity, monoclonal antibody is advantageous, because they can be synthesized without being polluted by other antibody.Modifier is " single Clone " refers to the characteristic of the antibody of the substantially homogeneous population obtained from antibody, and is not construed as needing through any spy Method is determined to produce antibody.For example, the hybridoma side described for the first time by Kohler et al (1975) Nature 256:495 Method can be prepared, or can be prepared monoclonal used according to the invention by DNA recombination method (referring to US 4816567) and be resisted Body.Also Clackson et al (1991) Nature, 352:624-628 can be used；Marks et al(1991) J.Mol.Biol., technology described in 222:581-597 is from phage antibody library or from carrying complete human immunoglobulin system The transgenic mice (Lonberg (2008) Curr.Opinion 20 (4): 450-459) of system separates monoclonal antibody.
The monoclonal antibody of this paper definitely includes " chimeric " antibody, wherein the part of heavy chain and/or light chain be originated from spy Corresponding sequence in the antibody of earnest kind is identical or homologous or belongs to specific specific antibodies classification or subclass, and the residue of chain Part is identical or homologous as the corresponding sequence in the antibody for being originated from another species or belongs to another antibody isotype or subclass, with And the segment of this antibody, as long as they show desired bioactivity (US 4816567；With Morrison et al (1984)Proc.Natl.Acad.Sci.USA,81:6851-6855).Chimeric antibody includes comprising dynamic from non-human primates The variable domains antigen-binding subsequences of object (for example, Old World Monkeys or ape and monkey) and human constant regions sequence it is " primatized " antibody.
" complete antibody " of this paper is comprising VL structural domain and VH structural domain and light chain constant domain (CL) and heavy chain The antibody of constant domain CH1, CH2 and CH3.Constant domain can be natural sequence constant domains (for example, the mankind day Right sequence constant domains) or its amino acid sequence variation.Complete antibody can have one or more " effector functions ", It refers to the bioactivity as caused by the region Fc (native sequence Fc region or the region amino acid sequence variation Fc) of antibody.Antibody The example of effector function includes that C1q is combined；The cytotoxicity of Complement Dependent；Fc receptor combines；Antibody-dependant it is cell-mediated Cytotoxicity (ADCC)；Phagocytosis；With the downward of cell surface receptor (such as B-cell receptor and BCR).
Complete antibody can be divided into different " classes by the amino acid sequence of the constant domain depending on their heavy chain Not ".There are 5 kinds of primary categories of complete antibody: IgA, IgD, IgE, IgG and IgM, and some in these can be further It is divided into " subclass " (isotype), for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2.Corresponding to the different classes of of antibody Heavy chain constant domain be referred to as α, δ, ε, γ and μ.The different classes of sub-unit structure of immunoglobulin and three kinds of rulers Very little configuration is well known.
The modification of antibody
Antibody disclosed herein can be modified.For example, them is made to be less immunogenicity to human experimenter.This can be with It is realized using any one of multinomial technology well known to those skilled in the art.It has been described more particularly below in these technologies It is some.
Humanization
The technology for reducing the vivo immunogenicity of non-human antibody or antibody fragment includes being referred to as those of " humanization ".
" humanized antibody " refers to the polypeptide of the Variable Area at least partly modified comprising human antibodies, wherein part Variable Area, be preferably significantly less than the part of complete human variable-domain, by pair from non-human species Sequence substitutions are answered, and wherein, the Variable Area of modification is connected at least another part of another protein, and the preferably mankind are anti- The constant region of body.Stating " humanized antibody " includes human antibodies, wherein one or more complementary determining region (" CDR ") amino Sour residue and/or one or more framework region (" FW " or " FR ") amino acid residues are resisted from rodent or other non-humans The same function site (analogous) displacement in body.State " humanized antibody " also comprising immunoglobulin amino acid sequence or it Segment comprising the FR of the basic amino acid sequence with human immunoglobulin and basic with non-human immunoglobulin Amino acid sequence CDR.
Non-human (for example, murine) antibody of " humanization " form is comprising being originated from non-human immunoglobulin most The chimeric antibody of small sequence.Or see in another way, humanized antibody is also comprising replacing human sequence, coming in non-human The human antibodies of the selected sequence of (for example, murine) antibody.Humanized antibody may include from identical or different species Conservative amino acid displacement or non-natural residues, do not significantly change its combination and/or bioactivity.This antibody is to include The chimeric antibody of minmal sequence from non-human immunoglobulin.
There are a large amount of humanization technologies, including ' CDR transplanting ', ' M8003 line ', ' going to be immunized ', ' resurfacing ' (also claim For ' exterior trim (veneering) '), ' compound antibody ', ' mankind's chain content optimizes (Human String Content Optimisation it) ' is mixed with frame.
CDR transplanting
In the art, humanized antibody is human immunoglobulin (receptor antibody), wherein from the mutual of receptor antibody It mends and determines that the residue of area (CDR) is come from non-human species such as mouse, rat, camel, ox, goat or rabbit with desirable properties CDR (donor antibody) replace (in fact, non-human CDR by ' transplanting " on human framework).In some embodiments, the mankind Framework region (FR) residue of immunoglobulin is replaced by corresponding non-human residues (for example, this can occur as specific FR When residue is to antigen is combined with remarkable effect).
In addition, humanized antibody may be embodied in and cannot all find in receptor antibody or the CDR or Frame sequence of implantation Residue.These modifications are carried out further to improve and maximize antibody performance.Therefore, in general, humanized antibody will include At least one and two kinds of whole variable domains in one aspect, wherein all the high of hypervariable region ring or whole become Qu Huan corresponds to those of non-human immunoglobulin, and the area all or essentially all of FR is human immunoglobulin sequence Those of.Humanized antibody optionally will also include at least part or human immunity ball egg of constant region for immunoglobulin (Fc) White at least part.
M8003 line
This method is made up of: by the V of the given non-human antibody to defined epitope specificityHOr VLStructural domain and people Class VHOr VLLibraries Combinatorial and special mankind's V structure domain is selected for the antigen of interest.Then by the selected mankind VH with VL libraries Combinatorial is to generate complete mankind VHxVL combination.Nature Biotechnology(N.Y.)12,(1994)899- This method is described in 903.
Compound antibody
In the method, two or more sections (segment) combination of the amino acid sequence from human antibodies is existed In final antibody molecule.They are constructed and combining multiple mankind VH and VL section in assembly, which exists Human T cell epitopes are limited or avoided in the final area compound antibody V.When needed, by with avoiding replacing for t cell epitope Facilitate or encode the section in the area V of t cell epitope for segment exchange to limit or avoid t cell epitope.US2008/ This method is described in 0206239A1.
It goes to be immunized
This method is related to removing the mankind (or other second species) T cell from the area V of therapeutic antibodies (or other molecules) Epitope.For example, by comparing with MHC binding motif (" motif " database e.g., being stored in www.wehi.edu.au), point Analyse the presence of MHC class II binding motif in therapeutic antibodies V region sequence.Alternatively, it can be used such as Altuvia et al. The computational threads method of (J.Mol.Biol.249244-250 (1995)) design identifies MHC class II binding motif；In these methods In, the combination energy of they and MHC class II protein is tested for the continuous overlapping peptide from V region sequence.Then by the number It is combined according to the information in other sequences feature, which is related to successfully presenting peptide, such as amphipathic, Rothbard motif and group Knit the cleavage site of Cathepsin B He other processive enzymes.
When the t cell epitope of identified possible second species (for example, mankind), by changing one or more ammonia Base acid eliminates them.The amino acid of modification can also close on the primary or two of protein usually in t cell epitope itself The epitope (therefore, may be not close in primary structure) of level structure.Most commonly, change be by way of displacement, But in some cases, it is more appropriate to add or delete meeting for amino acid.
All changes may be implemented by DNA recombinant technique, allow to by using the method well established (as oriented Mutagenesis) final molecule prepared by the expression of recombinant host.It is also possible, however, to use protein chemistry or any other molecule change Change mode.
Resurfacing
This method is related to:
(a) by the threedimensional model of building non-human antibody variable region, non-human (for example, rodent) antibody is determined The conformational structure of (or its segment)；
(b) it can reach distribution by the x- ray of enough non-humans and human antibody variable domains heavy chain and light chain using opposite Crystal structure generates sequence alignment, to provide one group of heavy chain and light chain framework position, wherein aligned position is enough non-98% It is identical in human antibody heavy chain and light chain；
(c) non-human antibody to humanization is determined using the frame position group generated in step (b), one group of heavy chain and light The amino acid residue of chain surface exposure；
(d) amino acid residue of one group of heavy chain and light chain surface exposure, the amino are identified by human antibodies amino acid sequence Sour residue and the amino acid residue of the one group of surface exposure limited in step (c) are the most similar, wherein the weight from human antibodies Chain and light chain are naturally in pairs or cannot naturally in pairs；
(e) in the amino acid sequence to the non-human antibody of humanization, use in step (d) in identify one group of heavy chain and The amino acid of the one group of heavy chain and light chain surface exposure that limit in the radical amino acid replacement step (c) of light chain surface exposure is residual Base；
(f) threedimensional model of the variable region of the non-human antibody obtained by the specified displacement of step (e) is constructed；
(g) by comparing the threedimensional model constructed in step (a) and (f), identification step (c) or (d) in the group that identifies Any amino acid residue, any original of the amino acid residue in any residue of the complementary determining region to humanizing non-human antibodies In 5 angstroms of son；And
(h) any residue identified in step (g) is changed from human amino acid residues as original non-human amino acid Residue, to limit non-human antibody's humanization group of the amino acid residue of surface exposure；Condition is not need to be walked first Suddenly (a), but step (a) must be carried out before step (g).
Super humanized
This method is by non-human sequence and functional human reproduction's germ line genes pedigree (human germline gene Repertoire) compare.Selection encodes typical case (canonical) structure those of identical or closely related with non-human sequence Human gene.These selected genes in CDR with highest homology are elected to be FR donor.Finally, non-human CDR is moved It plants on these mankind FR.This method is described in 2005/079479 A2 of patent WO.
The optimization of mankind's chain content
This method by non-human (for example, mouse) sequence compared with the pedigree of human reproduction's germ line genes, and by difference Scoring is mankind's chain content (HSC) of the Quantitative Sequence in the level of possible MHC/T cell epitope.Then by making target sequence HSC maximize rather than target sequence humanization is generated to a variety of different humanization variants using global identity measurement (in Molecular Immunology, 44, describe in (2007) 1986-1998).
Frame mixing
It will be fused in the CDR frame of non-human antibody comprising all known heavy chains and light chain human reproduction's germ line genes frame In the pond cDNA of frame.Then humanized antibody is selected for example, by the antibody library of elutriation (panning) phage display.This Methods 36,43-60 is described in (2005).
Definition
Pharmaceutically acceptable cation
The example of pharmaceutically acceptable unit price and bivalent cation is discussed at Berge, et al., J.Pharm.Sci., 66,1-19 (1977), it is incorporated herein by reference.
Pharmaceutically acceptable cation can be inorganic cation or organic cation.
The example of pharmaceutically acceptable monovalent inorganic cation includes but is not limited to alkali metal ion such as Na+And K+.Pharmacy The example of upper acceptable divalent inorganic cations includes but is not limited to alkaline earth metal cation such as Ca2+And Mg2+.Pharmaceutically may be used The example of the organic cation of receiving includes but is not limited to ammonium ion (i.e. NH4 +) and ammonium ion (such as the NH that replaces3R+、NH2R2 +、NHR3 +、NR4 +).The example of some suitable substituted ammonium ions is derived from the ammonium ion of those of following substitution: ethamine, two Ethamine, dicyclohexyl amine, triethylamine, butylamine, ethylenediamine, ethanol amine, diethanol amine, piperazine, benzylamine, phenylbenzylamine, choline, Portugal's first Amine and tromethamine and amino acid, such as lysine and arginine.The example of common quaternary ammonium ion is N (CH3)4 +。
Substituent group
As used in this article, phrase " optionally substituted " be related to its can be it is unsubstituted or its can be it is substituted Female group.
Unless otherwise prescribed, as used in this article, term " substitution " is related to it with one or more substituent groups Female group.Term " substituent group " be herein defined as in a conventional sense using and refer to such chemical part, covalently It is connected to, or if applicable, condenses in female group.Various substituent groups are well-known, and are used for theirs The method for forming and being introduced to various female groups is also well-known.
The example for being described in more detail below substituent group.
C1-12Alkyl: as used in this article, term " C1-12Alkyl " is related to by from 1 to 12 carbon atom The carbon atom of hydrocarbon compound remove hydrogen atom monovalent moiety obtained, can be aliphatic series or alicyclic, and it can be with It is saturated or unsaturated (such as part is unsaturated, completely unsaturated).As used in this article, term " C1-4Alkane Base " is related to by removing hydrogen atom monovalent moiety obtained from the carbon atom of the hydrocarbon compound with 1 to 4 carbon atom, It can be aliphatic series or alicyclic, and it can be saturated or unsaturated (such as part is unsaturated, completely unsaturated). Therefore, term " alkyl " includes the subclass being discussed below: alkenyl, alkynyl, naphthenic base etc..
The example of saturated alkyl includes but is not limited to methyl (C1), ethyl (C2), propyl (C3), butyl (C4), amyl (C5)、 Hexyl (C6) and heptyl (C7)。
The example of saturated linear alkyl includes but is not limited to methyl (C1), ethyl (C2), n-propyl (C3), normal-butyl (C4)、 N-pentyl (amyl) (C5), n-hexyl (C6) and n-heptyl (C7)。
The example for being saturated branched alkyl includes isopropyl (C3), isobutyl group (C4), sec-butyl (C4), tert-butyl (C4), isoamyl Base (C5) and neopentyl (C5)。
C2-12Alkenyl: as used in this article, term " C2-12Alkenyl " is related to having one or more carbon-to-carbon double bonds Alkyl.
The example of unsaturated alkenyl includes but is not limited to vinyl (ethenyl) (vinyl, vinyl ,-CH=CH2)、1- Acrylic (- CH=CH-CH3), 2- acrylic (allyl ,-CH-CH=CH2), isopropenyl (1- methyl ethylene ,-C (CH3) =CH2), cyclobutenyl (C4), pentenyl (C5) and hexenyl (C6)。
C2-12Alkynyl: as used in this article, term " C2-12Alkynyl " is related to having one or more carbon-carbon triple bonds Alkyl.
The example of unsaturated alkynyl includes but is not limited to acetenyl (- C ≡ CH) and 2-propynyl (2-propynyl) (alkynes third Base (propargyl) ,-CH2-C≡CH)。
C3-12Naphthenic base: as used in this article, term " C3-12Naphthenic base " is related to and the alkyl of ring group；That is, logical It crosses from the alicyclic ring atom of cyclic hydrocarbon (carbocyclic ring) compound and removes hydrogen atom monovalent moiety obtained, which has 3 to 7 Carbon atom, including 3 to 7 annular atoms.
The example of naphthenic base includes but is not limited to those naphthenic base, is originated from:
It is saturated monocycle hydrocarbon compound；
Cyclopropane (C3), cyclobutane (C4), pentamethylene (C5), hexamethylene (C6), cycloheptane (C7), methyl cyclopropane (C4)、 Dimethylcyclopropane (C5), methyl cyclobutane (C5), dimethylcyclobutane (C6), methyl cyclopentane (C6), dimethylcyclopentane (C7) and hexahydrotoluene (C7)；
Unsaturated monocyclic hydrocarbon compound；
Cyclopropylene (C3), cyclobutane (C4), cyclopentene (C5), cyclohexene (C6), methyl cyclopropene (C4), dimethylcyclopropene (C5), methyl cyclobutane (C5), dimethyl cyclobutane (C6), methyl cyclopentene (C6), dimethylcyclopentene (C7) and methyl cyclohexane Alkene (C7)；And
It is saturated polycyclic hydrocarbon compounds:
Norcarane (norcarane) (C7), norpinane (norpinane) (C7), norcamphane (norbornane, norbornane)(C7)。
C3-20Heterocycle: as used in this article, term " C3-20Heterocycle " is related to through the ring from heterocyclic compound Atom removes hydrogen atom monovalent moiety obtained, which has 3 to 20 annular atoms, wherein 1 to 10 is ring hetero atom. Preferably, each ring has 3 to 7 annular atoms, wherein 1 to 4 is ring hetero atom.
In this context, prefix (such as C3-20、C3-7、C5-6, etc.) indicate the number of annular atom or the number of annular atom Range, but regardless of being carbon atom or hetero atom.For example, as used in this article, term " C5-6Heterocycle " is related to having The heterocycle of 5 or 6 annular atoms.
The example of monocyclic heterocycles base includes but is not limited to be originated from those of following monocyclic heterocycles base:
N1: aziridine (C3), azetidine (C4), pyrrolidines (nafoxidine) (C5), pyrrolin is (for example, 3- pyrroles Quinoline, 2,5- pyrrolin) (C5), 2H- pyrroles or 3H- pyrroles (different pyrroles, isoxazole) (C5), piperidines (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine(C7)；
O1: oxirane (oxirane) (C3), oxetanes (oxetane) (C4), tetrahydrofuran (tetrahydro furan Mutter) (C5), oxole (divinylene oxide, oxole) (dihydrofuran) (C5), oxinane (exane) (oxinane) (C6), dihydropyran (C6), pyrans (C6), oxa-(disliking heptan, oxepin) (C7)；
S1: thiirane (C3), Thietane (C4), tiacyclopentane (thiophane) (C5), thia hexamethylene (tetrahydric thiapyran) (C6), thia cycloheptane (thiepane) (C7)；
O2: dioxolane (C5), dioxane (C6) and Dioxepane (dioxepane) (C7)；
O3: trioxane (C6)；
N2: imidazolidine (C5), pyrazolidine (two azo alkane) (C5), imidazoline (C5), pyrazoline (pyrazoline) (C5), piperazine (C6)；
N1O1: tetrahydro oxazole (C5), dihydro-oxazole (C5), tetrahydro isoxazole (C5), dihydro-isoxazole (C5), morpholine (C6)、 Tetrahydro oxazines (C6), dihydro oxazines (C6), oxazines (C6)；
N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6)；
N2O1: oxadiazines (C6)；
O1S1: oxygen dithiole (disliking thiophene, oxathiole) (C5) and thioxane (thiophene oxane) (C6)；With And
N1O1S1: dislike thiazine (C6)。
The example of substituted monocyclic heterocycles base includes the monocyclic heterocycles base that those replace, and is originated from the carbohydrate of loop type, example Such as, furanose (C5), such as arabinofuranose, lysol furanose (lyxofuranose), ribofuranose and furyl xylose (xylofuranse) and pyranose (C6), such as other pyranose (allopyranose), pyrans altrose, glucopyranose, Mannopyranose, pyrans gulose (gulopyranose), pyrans idose (idopyranose), galactopyranose and pyrans Talose (talopyranose).
C5-20Aryl: as used in this article, term " C5-20Aryl " is related to former by the aromatic ring from aromatic compounds Son removal hydrogen atom monovalent moiety obtained, has 3 to 20 annular atoms.As used in this article, term " C5-7 Aryl " is related to having 5 to 7 rings by removing hydrogen atom monovalent moiety obtained from the aromatic ring atom of aromatic compounds Atom, and as used in this article, term " C5-10Aryl " is related to by removing from the aromatic ring atom of aromatic compounds Hydrogen atom monovalent moiety obtained has 5 to 10 annular atoms.Preferably, each ring has 5 to 7 annular atoms.
In this context, prefix (such as C3-20、C5-7、C5-6、C5-10Deng) indicate annular atom (carbon atom or hetero atom) The range of number or the number of annular atom.For example, as used in this article, term " C5-6Aryl " is related to having 5 or 6 The aryl of annular atom.
Annular atom can all be carbon atom, such as in " carbon aryl ".
The example of carbon aryl includes but is not limited to be originated from those of following carbon aryl: benzene (i.e. phenyl) (C6), naphthalene (C10), Azulene (azulenes) (C10), anthracene (C14), phenanthrene (C14), naphthonaphthalene (C18) and pyrene (C16)。
The example of aryl comprising condensed ring (at least one its is aromatic ring) includes but is not limited to be originated from group below: indane (such as 2,3- dihydro -1H- indenes) (C9), indenes (C9), different indenes (C9), tetrahydronaphthalene (1,2,3,4- naphthane (C10), acenaphthene (C12)、 Fluorenes (C13), non-that alkene (C13), vinegar phenanthrene (C15) and aceanthrene (C16)。
Alternatively, annular atom may include one or more hetero atoms, such as in " heteroaryl ".The reality of bicyclic heteroaryl Example includes but is not limited to from those of following:
N1: pyrroles (Pyrrole) (azoles, azole) (C5), pyridine (azine) (C6)；
O1: furans (divinylene oxide, oxole) (C5)；
S1: thiophene (thiophene) (thiophene, thiole) (C5)；
N1O1: oxazole (C5), isoxazole (C5), isooxazine (C6)；
N2O1: oxadiazoles (furazan) (C5)；
N3O1: dislike triazole (C5)；
N1S1: thiazole (C5), isothiazole (C5)；
N2: imidazoles (1,3- diazole) (C5), pyrazoles (1,2- diazole) (C5), pyridazine (1,2- diazine) (C6), pyrimidine (1,3- Diazine) (C6) (for example, cytimidine, thymidine, uracil), pyrazine (1,4-diazines) (C6)；
N3: triazole (C5), triazine (C6)；And
N4: tetrazolium (C5)。
The example of heteroaryl comprising condensed ring includes but is not limited to:
From C below9(there are 2 condensed ring): benzofuran (O1), isobenzofuran (O1), indoles (N1), iso-indoles (N1), benzazole (N1), indoline (N1), isoindoline (N1), purine (N4) (for example, adenine, guanine), benzimidazole (N2), indazole (N2), benzoxazoles (N1O1), benzo isoxazole (N1O1), benzo dioxolen (benzo two dislikes cyclopentadienyl) (O2), benzofuraxan (N2O1), benzotriazole (N3), benzothiophene (S1), benzothiazole (N1S1), diazosulfide (N2S)；
From C below10(there are 2 condensed ring): chromene (O1), heterochromatic alkene (O1), chroman (chroman) (O1), it is heterochromatic full (O1), benzdioxan (O2), quinoline (N1), isoquinolin (N1), quinolizine (N1), benzoxazine (N1O1), benzodiazine (N2), pyrrole Pyridine and pyridine (N2), quinoxaline (N2), quinazoline (N2), cinnolines (N2), phthalazines (phthalazine) (N2), naphthyridines (diaza Naphthalene, naphthyridine) (N2), pteridine (N4)；
From C below11(there are 2 condensed ring): benzodiazepine(N2)；
From C below13(there are 3 condensed ring): carbazole (N1), dibenzofurans (O1), dibenzothiophenes (S1), carboline (N2), pyridine (perimidine, perimidine) (N pah2), pyridine diindyl (N2)；And
From C below14(there are 3 condensed ring): acridine (N1), xanthene (xanthene) (O1), thioxanthene (S1), dislike anthracene (oxanthrene)(O2), phenoxazine thiophene (phenoxathiin) (O1S1), azophenlyene (N2), phenoxazine (N1O1), phenthazine (N1S1)、 Thianthrene (S2), phenanthridines (N1), phenanthroline (N2), azophenlyene (N2)。
Above-mentioned group, no matter individually or a part of another substituent group, can itself optionally by selected from them this One or more groups of body and the other substituent group being listed below replace.
Halogen :-F ,-Cl ,-Br and-I.
Hydroxyl :-OH.
Ether :-OR, wherein R is ether substituent group, for example, C1-7Alkyl (is also known as C1-7Alkoxy is discussed below), C3-20Heterocycle (is also known as C3-20Heterocyclic oxy group) or C5-20Aryl (is also known as C5-20Aryloxy group), preferably C1-7Alkyl.
Alkoxy :-OR, wherein R is alkyl, for example, C1-7Alkyl.C1-7The example of alkoxy includes but is not limited to-OMe (methoxyl group) ,-OEt (ethyoxyl) ,-O (nPr) (positive propoxy) ,-O (iPr) (isopropoxy) ,-O (nBu) (n-butoxy) ,- O (sBu) (sec-butoxy) ,-O (iBu) (isobutoxy) and-O (tBu) (tert-butoxy).
Acetal :-CH (OR1)(OR2), wherein R1And R2It is independently acetal substitutent group, for example, C1-7Alkyl, C3-20Heterocycle Or C5-20Aryl, preferably C1-7Alkyl, alternatively, in the case where " ring-type " acetal groups, R1And R2Together with they connected two A oxygen atom and the carbon atom that they are connected are formed together the heterocycle with 4 to 8 annular atoms.The example of acetal groups includes But it is not limited to-CH (OMe)2、-CH(OEt)2With-CH (OMe) (OEt).
Hemiacetal :-CH (OH) (OR1), wherein R1It is hemiacetal substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20 Aryl, preferably C1-7Alkyl.The example of hemiacetal group includes but is not limited to-CH (OH) (OMe) and-CH (OH) (OEt).
Ketal :-CR (OR1)(OR2), wherein R1And R2It is as defined by acetal and R is contracting in addition to hydrogen Ketone substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of ketal group includes but not It is limited to-C (Me) (OMe)2、-C(Me)(OEt)2、-C(Me)(OMe)(OEt)、-C(Et)(OMe)2、-C(Et)(OEt)2With-C (Et)(OMe)(OEt)。
Hemiketal :-CR (OH) (OR1), wherein R1It is to be limited as being directed to hemiacetal, and R is hemiketal in addition to hydrogen Substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of hemiacetal group includes but not It is limited to-C (Me) (OH) (OMe) ,-C (Et) (OH) (OMe) ,-C (Me) (OH) (OEt) and-C (Et) (OH) (OEt).
Oxo (ketone group, -one) :=O.
Thioketones (Thione) (thio ketone, thioketone) :=S.
Imino group (imines) :=NR, wherein R is imino group substituent group, for example, hydrogen, C1-7Alkyl, C3-20Heterocycle or C5-20 Aryl, preferably hydrogen or C1-7Alkyl.The example of ester group includes but is not limited to=NH ,=NMe ,=Net and=NPh.
Formoxyl (formaldehyde, carbaldehyde, carboxaldehyde) :-C (=O) H.
Acyl group (ketone group) :-C (=O) R, wherein R is acyl substituent, for example, C1-7Alkyl (is also known as C1-7Alkane acyl Base), C3-20Heterocycle (is also known as C3-20Heterocyclic acyl) or C5-20Aryl (is also known as C5-20Aroyl), preferably C1-7Alkane Base.The example of acyl group includes but is not limited to-C (=O) CH3(acetyl group) ,-C (=O) CH2CH3(propiono) ,-C (=O) C (CH3)3(tertiary bytyry) and-C (=O) Ph (benzoyl, benzophenone).
Carboxyl (carboxylic acid) :-C (=O) OH.
Thiocarboxyl group (thiocarboxylic acid) :-C (=S) SH.
Sulfydryl for carboxyl (mercaptan carboxyl, Thiolocarboxy) (sulfydryl for carboxylic acid (thiol carboxylic acid), Thiolocarboxylic acid) :-C (=O) SH.
Thion carboxyl (thiono carboxylic acid) :-C (=S) OH.
Imidic acid (Imidic acid) :-C (=NH) OH.
Hydroxamic acid :-C (=NOH) OH.
Ester (carboxylate (carboxylate, carboxylic acid ester), Epoxide carbonyl (oxycarbonyl)) :- C (=O) OR, wherein R is ester substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.Ester group Example includes but is not limited to-C (=O) OCH3,-C (=O) OCH2CH3,-C (=O) OC (CH3)3With-C (=O) Oph.
Acyloxy (reversed ester) :-OC (=O) R, wherein R is acyloxy substituent group, for example, C1-7Alkyl, C3-20Heterocycle Or C5-20Aryl, preferably C1-7Alkyl.The example of acyloxy includes but is not limited to-OC (=O) CH3(acetoxyl group) ,-OC (=O) CH2CH3,-OC (=O) C (CH3)3,-OC (=O) Ph and-OC (=O) CH2Ph。
Oxygroup carbonyloxy group (Oxycarboyloxy) :-OC (=O) OR, wherein R is ester substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of ester group includes but is not limited to-OC (=O) OCH3,-OC (=O) OCH2CH3,-OC (=O) OC (CH3)3With-OC (=O) Oph.
Amino :-NR1R2, wherein R1And R2It is independently amino-substituent, for example, hydrogen, C1-7Alkyl (is also known as C1-7Alkane Base amino or two C1-7Alkyl amino), C3-20Heterocycle or C5-20Aryl, preferably H or C1-7Alkyl, alternatively, in " ring-type " amino In the case of, R1And R2The nitrogen-atoms connected together with them is formed together the heterocycle with 4 to 8 annular atoms.Amino can be primary Amino (- NH2), secondary amino group (- NHR1) or tertiary amino (- NHR1R2), and in cationic form, can be quaternary ammonium (-+ NR1R2R3).The example of amino includes but is not limited to-NH2、-NHCH3、-NHC(CH3)2、-N(CH3)2、-N(CH2CH3)2With- NHPh.The example of cyclic amino include but is not limited to '-aziridino, azetidinyl, pyrrolidines simultaneously, piperidino (piperidino), Piperazino (piperazino), morpholino and thiomorpholine generation.
Acylamino- (carbamoyl, carbamyl, amino carbonyl, formamide) :-C (=O) NR1R2, wherein R1And R2Solely It is on the spot amino-substituent, as defined by amino.The example of acylamino- includes but is not limited to-C (=O) NH2,-C (= O)NHCH3,-C (=O) N (CH3)2,-C (=O) NHCH2CH3With-C (=O) N (CH2CH3)2And acylamino-, wherein R1And R2 The nitrogen-atoms connected together with them is formed together heterocycle structure, such as exists, for example, piperidino carbonyl, morpholino carbonyl, thio In morpholino carbonyl and Piperazino carbonyl.
Thio acylamino (thiocarbamoyl) :-C (=S) NR1R2, wherein R1And R2It is independently amino-substituent, As for defined by amino.The example of acylamino- includes but is not limited to-C (=S) NH2,-C (=S) NHCH3,-C (=S) N (CH3)2With-C (=S) NHCH2CH3。
Acylamido (acylamino-) :-NR1C (=O) R2, wherein R1It is amide substituents, for example, hydrogen, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably hydrogen or C1-7Alkyl and R2It is acyl substituent, for example, C1-7Alkyl, C3-20Heterocycle Base or C5-20Aryl, preferably hydrogen or C1-7Alkyl.The example of amide group includes but is not limited to-NHC (=O) CH3,-NHC (=O) CH2CH3With-NHC (=O) Ph.R1And R2It can be formed together cyclic structure, such as existed, for example, succinimido, maleimide In amido and phthalimidyl:
Amino carbonyloxy group :-OC (=O) NR1R2, wherein R1And R2It is independently amino-substituent, as limited for amino 's.The example of amino carbonyloxy group includes but is not limited to-OC (=O) NH2,-OC (=O) NHMe ,-OC (=O) NMe2With-OC (=O) NEt2。
Urea groups :-N (R1)CONR2R3, wherein R2And R3It is independently amino-substituent, as defined by amino, and R1It is Carbamido substituted base, for example, hydrogen, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably hydrogen or C1-7Alkyl.The example of urea groups Including but not limited to-NHCONH2、-NHCONHMe、-NHCONHEt、-NHCONMe2、-NHCONEt2、-NMeCONH2、- NMeCONHMe、-NMeCONHEt、-NMeCONMe2With-NMeCONEt2。
Guanidine radicals :-NH-C (=NH) NH2。
Tetrazole radical: five yuan of aromatic rings with 4 nitrogen-atoms and a carbon atom,
Imino group :=NR, wherein R is imino group substituent group, for example, hydrogen, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, It is preferred that H or C1-7Alkyl.The example of imino group includes but is not limited to=NH ,=NMe and=NEt.
Amidine (amidino groups) :-C (=NR) NR2, wherein each R is amidine substituent group, for example, hydrogen, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably H or C1-7Alkyl.The example of amidine group includes but is not limited to-C (=NH) NH2,-C (=NH) NMe2With-C (=NMe) NMe2。
Nitro :-NO2。
Nitroso :-NO.
Azido :-N3。
Cyano (nitrile, nitrile, carbonitrile) :-CN.
Isocyano group :-NC.
Cyanato (Cyanato) :-OCN.
Isocyanato :-NCO.
Thiocyanogen (Thiocyano) (thiocyanogen) :-SCN.
Isothiocyano (isothiocyano) (isothiocyano, isothiocyanato) :-NCS.
Sulfydryl (Sulfhydryl) (mercaptan, thiol；Sulfydryl, mercapto) :-SH.
Thioether (sulfide) :-SR, wherein R is thioether substituent, for example, C1-7Alkyl (is also known as C1-7Alkylthio group), C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.C1-7The example of alkylthio group includes but is not limited to-SCH3With-SCH2CH3。
Disulphide :-SS-R, wherein R is disulphide substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Virtue Base, preferably C1-7Alkyl (also referred to as C1-7Alkyl disulfide).C1-7The example of alkyl disulfide group include but It is not limited to-SSCH3With-SSCH2CH3。
Sulfime base (sulfonium compound, sulfine) (sulfinyl, sulfoxide) :-S (=O) R, wherein R is sulfime base substituent group, example Such as, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of sulfime base group including but not limited to-S (= O)CH3With-S (=O) CH2CH3。
Sulfone (sulfonyl) :-S (=O)2R, wherein R is sulfone substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Virtue Base, preferably C1-7Alkyl, including, for example, fluorination or perfluorinated C1-7Alkyl.The example of sulfone group includes but is not limited to-S (=O)2CH3(mesyl) ,-S (=O)2CF3(trifyl, triflyl) ,-S (=O)2CH2CH3(ethylsulfonyl, Esyl) ,-S (=O)2C4F9(nine fluorine fourth sulfonyls, nonaflyl) ,-S (=O)2CH2CF3(trifluoro ethylsulfonyl, tresyl) ,- S (=O)2CH2CH2NH2(tauryl-, tauryl) ,-S (=O)2Ph (benzene sulfonyl, benzenesulfonyl (besyl)), 4- methylbenzene sulphur Acyl (tosyl (tosyl)), 4- chlorobenzenesulfonyl (chlorobenzenesulfonyl (closyl)), 4- bromophenylsulfonyl (bromobenzenesulfonyl (brosyl)), 4- nitrobenzophenone (nitrobenzenesulfonyl (nosyl)), 2- napsylate (naphthalene sulfonyl base, napsyl) and 5- diformazan Base amino-naphthalene -1- base sulphonic acid ester (dansyl (dansyl)).
Sulfinic acid (sulfino) :-S (=O) OH ,-SO2H。
Sulfonic acid (sulfo group) :-S (=O)2OH、-SO3H。
Sulfinic acid ester (sulfinate) (sulfinic acid ester, sulfinic acid ester) :-S (=O) OR；Wherein R is sub- Sulfonate substituent, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of sulfinate groups Including but not limited to-S (=O) OCH3(methoxyl group sulfinyl；Sulfinic acid methyl esters) and-S (=O) OCH2CH3(ethyoxyl sulfurous Acyl group；Sulfinic acid ethyl ester).
Sulphonic acid ester (sulfonate) (sulphonic acid ester, sulfonic acid ester) :-S (=O)2OR, wherein R is sulfonic acid Ester substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of sulfonate ester group include but It is not limited to-S (=O)2OCH3(methoxysulfonyl；Methylmesylate) and-S (=O)2OCH2CH3(ethoxysulfonyl；Sulfonic acid second Ester).
Sulfenyl oxygroup :-OS (=O) R, wherein R is thionyl oxy substituents, for example, C1-7Alkyl, C3-20Heterocycle Or C5-20Aryl, preferably C1-7Alkyl.The example of sulfurous acyloxy includes but is not limited to-OS (=O) CH3With-OS (=O) CH2CH3。
Sulfonyloxy :-OS (=O)2R, wherein R is sulfonyloxy substituent, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.The example of sulfonyloxy includes but is not limited to-OS (=O)2CH3(methanesulfonates) and-OS (= O)2CH2CH3(esilate).
Sulfuric ester :-OS (=O)2OR；Wherein R is sulfuric ester substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Virtue Base, preferably C1-7Alkyl.The example of sulfate group includes but is not limited to-OS (=O)2OCH3With-SO (=O)2OCH2CH3。
Sulfonamides (sulfamyl) (sulfonamides (sulfamoyl)；Sulfinic acid amide；Sulfenamide) :-S (=O) NR1R2, Wherein R1And R2It is independently amino-substituent, as defined by amino.The example of sulfonamides including but not limited to-S (= O)NH2,-S (=O) NH (CH3) ,-S (=O) N (CH3)2,-S (=O) NH (CH2CH3) ,-S (=O) N (CH2CH3)2With-S (=O) NHPh。
Sulfonamido (sulfonimide groups；Sulfonic acid amides；Sulfonamides) :-S (=O)2NR1R2, wherein R1And R2It is independently Amino-substituent, as defined by amino.The example of sulfonamido includes but is not limited to-S (=O)2NH2,-S (=O)2NH(CH3) ,-S (=O)2N(CH3)2,-S (=O)2NH(CH2CH3) ,-S (=O)2N(CH2CH3)2With-S (=O)2NHPh。
Sulfoamino-group :-NR1S (=O)2OH, wherein R1It is amino-substituent, as defined by amino.The reality of sulfoamino-group Example includes but is not limited to-NHS (=O)2OH and-N (CH3) S (=O)2OH。
Sulfonamido (Sulfonamino) :-NR1S (=O)2R, wherein R1It is amino-substituent, as limited for amino And R be sulfonamide substituent, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.Sulphonyl ammonia The example of base includes but is not limited to-NHS (=O)2CH3With-N (CH3) S (=O)2C6H5。
Sulfonamido (Sulfinamino) :-NR1S (=O) R, wherein R1It is amino-substituent, as limited for amino Fixed, and R is sulfinamino substituent, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl.It is sub- The example of sulfonamido includes but is not limited to-NHS (=O) CH3With-N (CH3) S (=O) C6H5。
Phosphino- (phosphine) :-PR2, wherein R is phosphino- substituent group, for example,-H, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, It is preferred that-H, C1-7Alkyl or C5-20Aryl.The example of phosphino- includes but is not limited to-PH2、-P(CH3)2、-P(CH2CH3)2、-P(t- Bu)2With-P (Ph)2。
Phospho :-P (=O)2。
Phosphoryl (phosphine oxide) :-P (=O) R2, wherein R is phosphinyl substituent group, for example, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably C1-7Alkyl or C5-20Aryl.The example of phosphoryl includes but is not limited to-P (=O) (CH3)2,-P (=O) (CH2CH3)2,-P (=O) (t-Bu)2With-P (=O) (Ph)2。
Phosphonic acids (phosphono) :-P (=O) (OH)2。
Phosphonate ester (phosphono base ester) :-P (=O) (OR)2, wherein R is phosphonate substituted base, for example,-H, C1-7Alkyl, C3-20 Heterocycle or C5-20Aryl, preferably-H, C1-7Alkyl or C5-20Aryl.The example of phosphonate groups includes but is not limited to-P (=O) (OCH3)2,-P (=O) (OCH2CH3)2,-P (=O) (O-t-Bu)2With-P (=O) (OPh)2。
Phosphoric acid (phosphonato) :-OP (=O) (OH)2。
Phosphate (phosphonato ester) :-OP (=O) (OR)2, wherein R is phosphate ester substituents, for example,-H, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably-H, C1-7Alkyl or C5-20Aryl.The example of bound phosphate groups includes but is not limited to-OP (=O) (OCH3)2,-OP (=O) (OCH2CH3)2,-OP (=O) (O-t-Bu)2With-OP (=O) (OPh)2。
Phosphorous acid :-OP (OH)2。
Phosphite ester :-OP (OR)2, wherein R is phosphite ester substituent group, for example,-H, C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably-H, C1-7Alkyl or C5-20Aryl.The example of phosphite group includes but is not limited to-OP (OCH3)2、-OP (OCH2CH3)2、-OP(O-t-Bu)2With-OP (OPh)2。
Phosphoramidite :-OP (OR1)-NR2 2, wherein R1And R2It is phosphoramidite substituent group, for example,-H, (optionally substituted) C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably-H, C1-7Alkyl or C5-20Aryl.The example of phosphoramidite group include but It is not limited to-OP (OCH2CH3)-N(CH3)2、-OP(OCH2CH3)-N(i-Pr)2With-OP (OCH2CH2CN)-N(i-Pr)2。
Phosphoramidate :-OP (=O) (OR1)-NR2 2, wherein R1And R2Phosphoramidate substituent group, for example,-H, (can Choose generation) C1-7Alkyl, C3-20Heterocycle or C5-20Aryl, preferably-H, C1-7Alkyl or C5-20Aryl.Phosphoramidic acid ester group Example include but is not limited to-OP (=O) (OCH2CH3)-N(CH3)2,-OP (=O) (OCH2CH3)-N(i-Pr)2With-OP (= O)(OCH2CH2CN)-N(i-Pr)2。
Alkylidene
C3-12Alkylidene: as used in this article, term " C3-12Alkylidene " is related to by from 3 to 12 carbon The identical carbon atoms of the hydrocarbon compound of atom (unless otherwise prescribed) remove two hydrogen atoms or each from two different carbon atoms A hydrogen atom two toothed portion obtained is removed, can be aliphatic series or alicyclic, and it can be saturation, part not It is saturation or complete unsaturated.Therefore, term " alkylidene " includes the following subclass being discussed below: alkenylene, alkynylene, ring Alkylidene etc..
Linear saturation C3-12The example of alkylidene includes but is not limited to-(CH2)n, wherein n is 3 to 12 integer, for example,- CH2CH2CH2(propylidene) ,-CH2CH2CH2CH2(butylidene) ,-CH2CH2CH2CH2CH2(pentylidene) and- CH2CH2CH2CH-2CH2CH2CH2(heptamethylene).
Branch is saturated C3-12The example of alkylidene includes but is not limited to-CH (CH3)CH2-、-CH(CH3)CH2CH2-、-CH (CH3)CH2CH2CH2-、-CH2CH(CH3)CH2-、-CH2CH(CH3)CH2CH2-、-CH(CH2CH3)-、-CH(CH2CH3)CH2And- CH2CH(CH2CH3)CH2-。
The unsaturated C of linear fraction3-12Alkylidene (C3-12Alkenylene and alkynylene) example include but is not limited to-CH= CH-CH2-、-CH2- CH=CH2,-CH=CH-CH2-CH2,-CH=CH-CH2-CH2-CH2,-CH=CH-CH=CH- ,-CH =CH-CH=CH-CH2,-CH=CH-CH=CH-CH2-CH2,-CH=CH-CH2- CH=CH- ,-CH=CH-CH2-CH2-CH =CH- and-CH2-C≡C-CH2-。
The unsaturated C of branched fraction3-12Alkylidene (C3-12Alkenylene and alkynylene) example include but is not limited to-C (CH3)=CH-.-C(CH3)=CH-CH2,-CH=CH-CH (CH3)-and-C ≡ C-CH (CH3)-。
Alicyclic saturation C3-12Alkylidene (C3-12Ring alkylidene) example include but is not limited to that (such as ring is amyl- for cyclopentylene 1,3- subunit) and cyclohexylidene (such as hexamethylene -1,4- subunit).
The unsaturated C of cycloaliphatic moiety3-12Alkylidene (C3-12Ring alkylidene) example include but is not limited to cyclopentenylidene (such as 4- cyclopentene -1,3- subunit), cyclohexadienylidene (such as 2- cyclohexene -1,4- subunit；3- cyclohexene -1,2- subunit；2, 5- cyclohexadiene -1,4- subunit).
Carbamate nitrogen-protecting group group: term " carbamate nitrogen-protecting group group " is related to such part, masking Nitrogen in imine linkage, and be well known in the art.These groups have a structure that
Wherein R '10It is R as defined above.Largely suitable group is described in Greene, T.W.and Wuts, G.M.,Protective Groups in Organic Synthesis,3rd Edition,John Wiley&Sons,Inc., 1999 page 503 to 549, it is incorporated herein by reference.
Hemiacetal amine nitrogen-protecting group group: term " hemiacetal amine nitrogen-protecting group group " is related to the group having following structure:
Wherein R '10It is R as defined above.The a large amount of suitable group as amide blocking group is described in Greene,T.W.and Wuts,G.M.,Protective Groups in Organic Synthesis,3rd Edition, Page 633 to 647 of John Wiley&Sons, Inc., 1999, it is incorporated herein by reference.
Group carbamate nitrogen-protecting group group and hemiacetal nitrogen-protecting group group can be commonly referred to as " the nitrogen for synthesis Blocking group ".
Conjugate
The present invention provides the conjugate of the PBD compound comprising being connected to antibody via connector unit.
In one embodiment, conjugate includes the antibody for being connected to introns linking group, is connected to initiator (trigger) introns, the initiator for being connected to selfdecomposition (from eliminating, self-destruction, self-immolative) connector and connection To the selfdecomposition connector of the position N10 of PBD compound.Following illustrate this conjugates:
Wherein, Ab is antibody defined above and PBD is Pyrrolobenzodiazepines described hereinCompound (D).Illustrate to show the R corresponding in certain embodiments of the inventionL’、A、L1And L2Part.RL’It can be RL1’Or RL2’.D is wherein to remove RL1’Or RL2’DL。
Purposes of the present invention suitable for PBD compound to be provided to preferred position subject.In preferred embodiment party In formula, conjugate allows to discharge the active PBD compound for not retaining any junction portion.There is no can influence PBD compound Reactivity residual.
Antibody is connected to PBD drug moiety D by one or more covalent bonds by connector.Connector is difunctionality or more officials The part of energy, can be used for connecting one or more drug moieties (D) and antibody units (Ab) to form antibody-drug conjugates (ADC).Connector (RL’) except cell (that is, extracellular) can be stable or it is by enzymatic activity, hydrolysis or other are new Old metabolic conditions are cleavable.It can be convenient using having for being bound to the connector of the reactive functional group degree of drug moiety and antibody Ground prepares antibody-drug conjugates (ADC).The cysteine mercaptan or amine of antibody (Ab), such as N-terminal or amino acid side chain are (such as Lysine), it can be with connector or introns reagent, PBD drug moiety (D) or agent-linker reagent (DL, D-RL) functional group Form key, wherein RLIt can be RL1Or RL2。
The connector of ADC is preferably prevented from the aggregation of ADC molecule, and ADC is kept to be soluble in aqueous medium and protected It holds in free state.
The connector of ADC is preferably extracellularly being stablized.Before conveying or being delivered to cell, antibody-drug conjugates (ADC) it is preferably stable and keeps complete, that is, antibody remains attached to drug moiety.Connector is steady outside target cell Fixed, and can be cut in the cell with effective speed.Effective connector is incited somebody to action: the specific binding characteristics of (i) holding antibody； (ii) allow the Intracellular delivery of conjugate or drug moiety；(iii) keep stable and complete, that is, until conjugate is delivered Or it is delivered to its target site and just cuts；And (iv) keeps cytotoxic effect, the killing functions of immunocytes of PBD drug moiety Or cyto-inhibition.The steady of ADC can be measured by standard analytical techniques such as mass spectrum, HPLC and separation/analytical technology LC/MS It is qualitative.
The covalent linkage of antibody and drug moiety needs connector tool, and there are two reactive functional groups, that is, in reaction meaning Divalent.It can be used for connecting two or more funtion parts or biologically-active moiety such as peptide, nucleic acid, drug, toxin, antibody, half to resist Former and reporter group bivalent linker is known, and has had method to describe the conjugate that they are obtained (Hermanson,G.T.(1996)Bioconjugate Techniques；Academic Press:New York,p 234- 242)。
In another embodiment, connector can be replaced by adjusting aggregation, solubility or reactive group.For example, Sulfonate substituent can increase the water solubility of reagent and promote the coupling reaction of linker reagents and antibody or drug moiety, Or promote Ab-L and DLOr DLThe coupling reaction of-L and Ab, this depends on preparing the synthesis path that ADC is used.
In one embodiment, L-RL’It is following group:
Wherein, asterisk indicates the point for being connected to drug unit (D), and Ab is antibody (L), L1It is connector, A is by L1It is connected to The linking group of antibody, L2Be covalent bond or with-OC (=O)-be formed together selfdecomposition connector and L1Or L2It is cleavable Connector.
L1Preferably cleavable connector, and can be by being known as the initiator for activating joint cutting.
When it is present, L1And L2Property can change extensively.Based on their Cutting feature come these groups of selection, It can be determined by the condition in the site that conjugate is delivered.Although also can be used through pH variation (for example, acid or alkali are not Stability), temperature or be cleavable connector by radiation (for example, to unstability of light), but cut by the effect of enzyme It is preferred for those of cutting connector.Cleavable connector under reduction or oxidizing condition can also be used in the present invention.
L1It may include the continuous sequence of amino acid.Amino acid sequence can be the target substrate cut for digestion, to permit Perhaps L-R is discharged from the position N10L’。
In one embodiment, L can be cut by the effect of enzyme1.In one embodiment, enzyme is esterase or peptide Enzyme.
In one embodiment, there are L2And selfdecomposition connector is formed together with-C (=O) O-.In an embodiment party In formula, L2It is the substrate for enzymatic activity, to allow to discharge L-R from the position N10L’。
In one embodiment, work as L1In the effect of enzyme be cleavable and there are L2When, L is cut in digestion1And L2Between Key.
L1And L2(when it is present) key connection selected from the following can be passed through:
- C (=O) NH-,
- C (=O) O-,
- NHC (=O)-,
- OC (=O)-,
- OC (=O) O-,
- NHC (=O) O-,
- OC (=O) NH- and
- NHC (=O) NH-.
It is connected to L2L1Amino group can be the end N- of amino acid, or amino acid side chain can be originated from and (such as rely ammonia Sour amino acid side chain) amino group.
It is connected to L2L1Carboxylic group can be the end C- of amino acid, or amino acid side chain (such as paddy ammonia can be originated from Sour amino acid side chain) carboxylic group.
It is connected to L2L1Hydroxyl group can be originated from amino acid side chain (such as serine amino acids side chain) hydroxyl base Group.
Term " amino acid side chain " includes those of discovery group in the following: (i) naturally occurring amino acid, such as the third ammonia Acid, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, Leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine； (ii) micro-amino acid (minor amino acids), such as ornithine and citrulling；(iii) unnatural amino acid, beta-amino The synthetic analogues and derivative of sour, naturally occurring amino acid；And (iv) all enantiomters, diastereoisomer, The isomer that is rich in, isotope labelling (for example,2H、3H、14C、15N), protected form and their racemic mixing Object.
In one embodiment ,-C (=O) O- and L2It is formed together following group:
Wherein, asterisk indicates the point for being connected to the position N10, and wave expression is connected to connector L1Point, Y be-N (H)-,- O- ,-C (=O) N (H)-or-C (=O) O-, and n is 0 to 3.Phenylene ring is optionally by one described herein, two Or three substituent groups replace.In one embodiment, subphenyl group is optionally by halogen, NO2, R or OR replace.
In one embodiment, Y is NH.
In one embodiment, n is 0 or 1.Preferably, n is 0.
When Y is NH and n is 0, selfdecomposition connector can be referred to as aminobenzyl carbonyl linker (PABC).
When distal site is activated, selfdecomposition connector is allowed to discharge shielded compound, along road as shown below Line carries out (n=0):
Wherein, L*It is the activated form of the remainder of connector.These groups have and make to activate site and shieldedization Close the advantage of object separation.As described above, subphenyl group can be optionally substituted.
In an embodiment as described herein, group L*It is connector L as described herein1, may include dipeptides Group.
In another embodiment ,-C (=O) O- and L2It is formed together group selected from the following:
Wherein, asterisk, wave, Y and n be as defined above.Each phenylene ring is optionally described herein One, two or three substituent group replaces.In one embodiment, the phenylene ring with Y substituent group is optionally substituted And the phenylene ring for not having Y substituent group is unsubstituted.In one embodiment, with the phenylene ring of Y substituent group Be it is unsubstituted and do not have Y substituent group phenylene ring be optionally substituted.
In another embodiment ,-C (=O) O- and L2It is formed together group selected from the following:
Wherein, asterisk, wave, Y and n are that as defined above, E is O, S or NR, and D is N, CH or CR, and F is N, CH or CR.
In one embodiment, D is N.
In one embodiment, D is CH.
In one embodiment, E is O or S.
In one embodiment, F is CH.
In a preferred embodiment, connector is cathepsin unstability connector.
In one embodiment, L1Including dipeptides.The dipeptides can be expressed as-NH-X1-X2- CO-, wherein-NH- and- CO- respectively indicates amino acid group X1And X2N and C-terminal.Amino acid in the dipeptides can be any group of natural amino acid It closes.When connector is cathepsin unstability connector, dipeptides can be the action site of the cutting of cathepsin mediation.
In addition, distinguishing such as Glu and Lys, CO for those of carboxyl or amino side chain degree of functionality amino acid group The side chain functionalities can be indicated with NH.
In one embodiment, dipeptides-NH-X1-X2Group-X in-CO-1-X2, it is selected from:
-Phe-Lys-、
-Val-Ala-、
-Val-Lys-、
-Ala-Lys-、
-Val-Cit-、
-Phe-Cit-、
-Leu-Cit-、
-Ile-Cit-、
-Phe-Arg-、
- Trp-Cit-,
Wherein, Cit is citrulling.
Preferably, dipeptides-NH-X1-X2Group-X in-CO-1-X2, it is selected from:
-Phe-Lys-、
-Val-Ala-、
-Val-Lys-、
-Ala-Lys-、
-Val-Cit-。
It is highly preferred that dipeptides-NH-X1-X2Group-X in-CO-1-X2, it is-Phe-Lys- or-Val-Ala-.
The combination of other dipeptides can be used comprising Dubowchik et al., Bioconjugate Chemistry, Those of 2002,13,855-869 descriptions, are incorporated by reference herein.
In one embodiment, in the appropriate case, amino acid side chain is derivative.For example, the ammonia of amino acid side chain Base group or carboxylic group can be derivative.In one embodiment, the amino group of side chain amino acid (such as lysine) NH2It is selected from by NHR and NRR ' derivative form of group that forms.In one embodiment, side chain amino acid (such as asparagus fern ammonia Acid) carboxylic group COOH be selected from by COOR, CONH2, CONHR and CONRR ' composition group derivative form.
In one embodiment, in the appropriate case, amino acid side chain is chemoproection.Side chain protecting group can be with It is below in relation to group RLThe group of discussion.Present inventor have determined that shielded amino acid sequence is by enzyme cleavable.Example Such as, it has been determined that the dipeptide sequence of the Lys residue comprising Boc side chain protection is histone enzyme cleavable.
Blocking group for amino acid side chain is well known in the art and retouches in Novabiochem catalogue It states.Other protecting groups are described in Protective Groups in Organic Synthesis, Greene and Wuts Group's strategy.
For shown below possible side chain protecting group with those of reactive side chain degree of functionality amino acid:
Arg:Z, Mtr, Tos；
Asn:Trt, Xan；
Asp:Bzl, t-Bu；
Cys:Acm, Bzl, Bzl-OMe, Bzl-Me, Trt；
Glu:Bzl, t-Bu；
Gln:Trt, Xan；
His:Boc, Dnp, Tos, Trt；
Lys:Boc, Z-Cl, Fmoc, Z, Alloc；
Ser:Bzl, TBDMS, TBDPS；
Thr:Bz；
Trp:Boc；
Tyr:Bzl, Z, Z-Br.
In one embodiment, when it is present, it selects Side chain protective group and is provided as end-capping group or partially end-blocked base The group of group is orthogonal.Therefore, remove side chain protecting group do not remove end-capping group or it is any be end-capping group a part guarantor Protect group functionalities.
In other embodiments of the invention, selected amino acid is that do not have those of reactive side chain degree of functionality. For example, amino acid can be selected from: Ala, Gly, Ile, Leu, Met, Phe, Pro and Val.
In one embodiment, dipeptides is used with selfdecomposition splice combinations.Selfdecomposition connector can connect to-X2-。
When there are selfdecomposition connector ,-X2It is connected directly to selfdecomposition connector.Preferably, group-X2- CO- is connected to Y, Wherein, Y is NH, to form group-X2-CO-NH-。
-NH-X1It is connected directly to A.A may include functional group-CO-, to be formed and-X1The amide of connection.
In one embodiment, L1And L2It together include group NH-X with-OC (=O)-1-X2-CO-PABC-.PABC base Group is connected directly to the position N10.Preferably, selfdecomposition connector and dipeptides are formed together group-NH-Phe-Lys-CO-NH- PABC-, described below:
Wherein, asterisk indicates that the point for being connected to the position N10 and wave expression are connected to connector L1Remainder Point or the point for being connected to A.Preferably, wave indicates the point for being connected to A.The side chain of Lys amino acid can be shielded, example Such as, it is protected with Boc, Fmoc or Alloc described above.
Alternatively, selfdecomposition connector and dipeptides are formed together group-NH-Val-Ala-CO-NH-PABC-, following It shows:
Wherein, asterisk and wave be as defined above.
Alternatively, selfdecomposition connector and dipeptides are formed together group-NH-Val-Cit-CO-NH-PABC-, following It shows:
Wherein, asterisk and wave be as defined above.
In one embodiment, A is covalent bond.Therefore, L1It is directly connected to antibody.For example, working as L1Comprising continuous Amino acid sequence when, the N-terminal of sequence can be directly connected to antibody.
Therefore, when A is covalent bond, antibody and L1Between connection can be selected from:
- C (=O) NH-,
- C (=O) O-,
- NHC (=O)-,
- OC (=O)-,
- OC (=O) O-,
- NHC (=O) O-,
- OC (=O) NH-,
- NHC (=O) NH-,
- C (=O) NHC (=O)-,
-S-、
-S-S-、
-CH2C (=O)-and
=N-NH-.
It is connected to the L of antibody1Amino group can be the N-terminal of amino acid, or amino acid side chain can be originated from and (such as relied Valine amino acid side chain) amino group.
It is connected to the L of antibody1Carboxylic group can be the C-terminal of amino acid, or amino acid side chain (such as paddy can be originated from Valine amino acid side chain) carboxylic group.
It is connected to the L of antibody1Hydroxyl group can be originated from amino acid side chain (such as serine amino acids side chain) hydroxyl Base group.
It is connected to the L of antibody1Thiol group can be originated from amino acid side chain (such as serine amino acids side chain) sulphur Alcohol groups.
Above with respect to L1Amino, carboxyl, hydroxyl and thiol group annotation also apply to antibody.
In one embodiment, L2It is indicated together with-OC (=O)-:
Wherein, asterisk indicates the point for being connected to the position N10, and wave expression is connected to L1Point, n is that 0 to 3, Y is covalent Key or functional group and E are can to activate group, such as by enzyme effect or light, to generate the unit of selfdecomposition.Phenylene ring Optionally it is further substituted with by one, two or three substituent group described herein.In one embodiment, phenylene base Group is optionally by halogen, NO2, R or OR be further substituted with.Preferably, n is 0 or 1, most preferably 0.
Selection E makes group be easy to activate, for example, activating by light or by the effect of enzyme.E can be-NO2Or grape Uronic acid.The former may may be vulnerable to the effect of GRD beta-glucuronidase vulnerable to the effect of nitroreductase, the latter.
In this embodiment, when E is activated, selfdecomposition connector will allow to discharge shielded compound, along with Route shown in lower carries out (n=0):
Wherein, asterisk indicates the point for being connected to the position N10, and E* is the activated form of E, and Y is as described above.These Group has the advantage for separating activation site with shielded compound.As described above, subphenyl group can be Optionally it is further substituted with.
Group Y can be down to L1Covalent bond.
Group Y can be functional group selected from the following:
- C (=O)-,
-NH-、
-O-、
- C (=O) NH-,
- C (=O) O-,
- NHC (=O)-,
- OC (=O)-,
- OC (=O) O-,
- NHC (=O) O-,
- OC (=O) NH-,
- NHC (=O) NH-,
- NHC (=O) NH,
- C (=O) NHC (=O)-and
-S-。
Work as L1When being dipeptides, preferably Y is-NH- or-C (=O)-, to form L1Amido bond between Y.At this In embodiment, dipeptide sequence needs not be the substrate of enzymatic activity.
In another embodiment, A is spacer groups.Therefore, L1It is indirectly connected with antibody.
L1Key connection selected from the following can be passed through with A:
- C (=O) NH-,
- C (=O) O-,
- NHC (=O)-,
- OC (=O)-,
- OC (=O) O-,
- NHC (=O) O-,
- OC (=O) NH- and
- NHC (=O) NH-.
In one embodiment, group A is:
Wherein, asterisk expression is connected to L1Point, wave indicates to be connected to the point of antibody, and n is 0 to 6.At one In embodiment, n is 5.
In one embodiment, group A is:
Wherein, asterisk expression is connected to L1Point, wave indicates to be connected to the point of antibody, and n is 0 to 6.At one In embodiment, n is 5.
In one embodiment, group A is:
Wherein, asterisk expression is connected to L1Point, wave indicates to be connected to the point of antibody, and n is 0 or 1, and m be 0 to 30.In one preferred embodiment, it is 0 to 10,1 to 8, preferably 4 to 8 and most preferably 4 or 8 that n, which is 1 and m,.? In another embodiment, m is 10 to 30, and preferably 20 to 30.Alternatively, m is 0 to 50.Preferably In, m is preferably 10-40, and n is 1.
In one embodiment, group A is:
Wherein, asterisk expression is connected to L1Point, wave indicates to be connected to the point of antibody, and n is 0 or 1, and m be 0 to 30.In one preferred embodiment, it is 0 to 10,1 to 8, preferably 4 to 8 and most preferably 4 or 8 that n, which is 1 and m,.? In another embodiment, m is 10 to 30, and preferably 20 to 30.Alternatively, m is 0 to 50.Preferably In, m is preferably 10-40, and n is 1.
In one embodiment, the connection between antibody and A passes through the thiol residue of antibody and the dimaleoyl imino of A Group.
In one embodiment, the connection between antibody and A is:
Wherein, asterisk indicates the point for being connected to the remainder of A, and wave indicates the remainder for being connected to antibody Point.In this embodiment, S atom generally originates from antibody.
In each of embodiment of above, alternative degree of functionality can be used instead of described below and be originated from Malaysia Imido group:
Wherein, wave indicates the point for being connected to antibody as before, and asterisk is indicated to the remainder of A group Key.
In one embodiment, the group from maleimide is replaced with following group:
Wherein, wave indicates the point for being connected to antibody, and asterisk indicates the key of the remainder to A group.
In one embodiment, the group from maleimide is replaced by group, the group optionally with antibody one It rises, is selected from:
- C (=O) NH-,
- C (=O) O-,
- NHC (=O)-,
- OC (=O)-,
- OC (=O) O-,
- NHC (=O) O-,
- OC (=O) NH-,
- NHC (=O) NH-,
- NHC (=O) NH,
- C (=O) NHC (=O)-,
-S-、
-S-S-、
-CH2C (=O)-,
- C (=O) CH2-、
=N-NH- and
- NH-N=.
In one embodiment, the group from maleimide is replaced by group, the group optionally with antibody one It rises, is selected from:
Wherein, wave indicates to be connected to the point of antibody or the key of the remainder to A group, and asterisk indicates connection To the key of the other point or the remainder to A group of antibody.
It describes and is suitable for L in WO 2005/0820231It is connected to the other groups of antibody.
In one embodiment, there are linking group A, and there are initiator L1And selfdecomposition connector L is not present2.Cause This, L1It is directly connected to drug unit via key.It is equal in this embodiment, L2It is key.Work as DLWhen being Formula II, this can be with It is especially interesting.
L1Key connection selected from the following can be passed through with D:
- C (=O) N <,
- C (=O) O-,
- NHC (=O)-,
- OC (=O)-,
- OC (=O) O-,
- NHC (=O) O-,
- OC (=O) N <, and
- NHC (=O) N <,
Wherein, N < or O- is a part of D.
In one embodiment, L1With D by key connection selected from the following:
- C (=O) N <, and
- NHC (=O)-.
In one embodiment, L1One end comprising dipeptides and dipeptides is connected to D.As described above, dipeptides In amino acid can be any combination of natural amino acid and unnatural amino acid.In some embodiments, dipeptides includes Natural amino acid.When connector is the unstable connector of cathepsin, dipeptides is the effect for the cutting that cathepsin mediates Site.Then dipeptides is the recognition site of cathepsin.
In one embodiment, dipeptides-NH-X1-X2Group-X in-CO-1-X2, it is selected from:
-Phe-Lys-、
-Val-Ala-、
-Val-Lys-、
-Ala-Lys-、
-Val-Cit-、
-Phe-Cit-、
-Leu-Cit-、
-Ile-Cit-、
- Phe-Arg- and
-Trp-Cit-；
Wherein, Cit is citrulling.In this dipeptides ,-NH- is X1Amino group, and CO is X2Carbonyl group.
Preferably, dipeptides-NH-X1-X2Group-X in-CO-1-X2, it is selected from:
-Phe-Lys-、
-Val-Ala-、
-Val-Lys-、
- Ala-Lys- and
-Val-Cit-。
It is highly preferred that dipeptides-NH-X1-X2Group-X in-CO-1-X2, it is-Phe-Lys- or-Val-Ala-.
Other interested dipeptides, which combine, includes:
-Gly-Gly-、
- Pro-Pro- and
-Val-Glu-。
The combination of other dipeptides, including those described above can be used.
In one embodiment, L1- D is:
Wherein ,-NH-X1-X2- CO is dipeptides, and a part of-N <be drug unit, asterisk expression is connected to drug unit The point of remainder, and wave expression is connected to L1Remainder point or be connected to the point of A.Preferably, wave Indicate the point for being connected to A.
In one embodiment, dipeptides is val-ala and L1- D is:
Wherein asterisk ,-N < and wave be as defined above.
In one embodiment, dipeptides is Phe-Lys and L1- D is:
Wherein asterisk ,-N < and wave be as defined above.
In one embodiment, dipeptides is valine-citrulline.
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, and n be 0 to 6.In one embodiment, n is 5.
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, and n be 0 to 6.In one embodiment, n is 5.
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, n is 0 or 1, and And m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 8, preferably 4 to 8, most preferably 4 or 8。
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, n is 0 or 1, and And m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 7, preferably 3 to 7, most preferably 3 or 7。
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, and n be 0 to 6.In one embodiment, n is 5.
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, and n be 0 to 6.In one embodiment, n is 5.
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, n is 0 or 1, and And m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 8, preferably 4 to 8, most preferably 4 or 8。
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, n is 0 or 1, and And m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 8, preferably 4 to 8, most preferably 4 or 8。
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, S are the methylthio groups with body unit, wave expression is connected to ligand The point of the rest part of unit, and n is 0 to 6.In one embodiment, n is 5.
In one embodiment, group A-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, S are the methylthio groups with body unit, wave expression is connected to ligand The point of the remainder of unit, and n is 0 to 6.In one embodiment, n is 5.
In one embodiment, group A1-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, S are the methylthio groups with body unit, wave expression is connected to ligand The point of the remainder of unit, n is 0 or 1, and m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 8, preferably 4 to 8, most preferably 4 or 8.
In one embodiment, group A1-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave expression are connected to the point with body unit, n is 0 or 1, and And m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 7, preferably 4 to 8, most preferably 4 or 8。
In one embodiment, group A1-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave indicate the point for being connected to the remainder with body unit, And n is 0 to 6.In one embodiment, n is 5.
In one embodiment, group A1-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave indicate the point for being connected to the remainder with body unit, And n is 0 to 6.In one embodiment, n is 5.
In one embodiment, group A1-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave indicate the point for being connected to the remainder with body unit, n It is 0 or 1, and m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 8, preferably 4 to 8, Most preferably 4 or 8.
In one embodiment, group A1-L1It is:
Wherein, asterisk expression is connected to L2Or the point of D, wave indicate the point for being connected to the remainder with body unit, n It is 0 or 1, and m is 0 to 30.In one preferred embodiment, n be 1 and m be 0 to 10,1 to 8, preferably 4 to 8, Most preferably 4 or 8.
Group RL’It may originate from group RL.By the way that antibody is connected to RLFunctional group can be by group RLIt is converted into group RL’.Other steps can be taken RLIt is converted into RL’.These steps may include removing existing blocking group or introducing suitably Functional group.
RL
Connector may include the peptide moiety of the albumen enzyme cleavable comprising one or more Amino Acid Units.Peptide can be passed through Solid phase known to chemical field or liquid-phase synthesis process (E. and K.Lübke,The Peptides,volume 1, pp 76-136 (1965) Academic Press), including t-BOC chemistry (Geiser et al " Automation of solid-phase peptide synthesis"in Macromolecular Sequencing and Synthesis,Alan R.Liss, Inc., 1988, pp.199-218) and Fmoc/HBTU chemistry (Fields, G.and Noble, R. (1990) " Solid phase peptide synthesis utilizing 9-fluoroenylmethoxycarbonyl amino acids", Int.J.Peptide Protein Res.35:161-214), in Fully automated synthesis instrument such as Rainin Symphony peptide synthesizer (Protein Technologies, Inc., Tucson, AZ) or Model 433 (Applied Biosystems, Foster City, CA) on prepare peptide linker reagent.
Illustrative Amino acid linker includes dipeptides, tripeptides, tetrapeptide or pentapeptide.Illustrative dipeptides includes: valine-melon The illustrative tripeptides of propylhomoserin (vc or val-cit), alanine-phenylalanine (af or ala-phe) includes: glycine-valine- Citrulling (gly-val-cit) and Gly-Gly-Gly (gly-gly-gly).Ammonia comprising Amino acid linker component Base acid residue includes those of naturally occurring and micro-amino acid and non-naturally occurring amino acid analogue, such as citrulling. The digestion by the relevant protease of certain enzyme such as tumour, cathepsin B, C and D or plasmase is cut at them Selectivity aspect, can design and optimize Amino acid linker.
Amino acid side chain includes those of naturally occurring and micro-amino acid and non-naturally occurring amino acid analogue, Such as citrulling.Amino acid side chain include hydrogen, methyl, isopropyl, isobutyl group, sec-butyl, benzyl, to hydroxybenzyl ,-CH2OH、- CH(OH)CH3、-CH2CH2SCH3、-CH2CONH2、-CH2COOH、-CH2CH2CONH2、-CH2CH2COOH、-(CH2)3NHC (=NH) NH2、-(CH2)3NH2、-(CH2)3NHCOCH3、-(CH2)3NHCHO、-(CH2)4NHC (=NH) NH2、-(CH2)4NH2、-(CH2)4NHCOCH3、-(CH2)4NHCHO、-(CH2)3NHCONH2、-(CH2)4NHCONH2、-CH2CH2CH(OH)CH2NH2, 2- pyridine first Base, 3- pyridylmethyl, 4- pyridylmethyl, phenyl, cyclohexyl and with flowering structure:
When amino acid side chain includes other than hydrogen (glycine), the carbon atom of amino acid side chain connection is chiral. Each carbon atom of amino acid side chain connection is independently (S) or (R) configuration or racemic mixture.Therefore, agent-linker Reagent can be enantiomer-pure, racemic or diastereomer.
In an exemplary embodiment, amino acid side chain is selected from those of natural and unnatural amino acid, including the third ammonia Acid, 2- amino -2- cyclohexyl-acetic acid, 2- amino -2- phenylacetic acid, arginine, asparagine, aspartic acid, cysteine, paddy Glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, nor-leucine, phenylpropyl alcohol ammonia Acid, proline, serine, threonine, tryptophan, tyrosine, valine, γ-aminobutyric acid, alpha, alpha-dimethyl gamma-amino fourth Acid, beta, beta-dimethyl γ-aminobutyric acid, ornithine and citrulling (Cit).
Introns with aminobenzyl carbamyl (PAB) selfdecomposition, suitable for constitute for being bound to antibody Connector-PBD drug moiety intermediate exemplary valine-citrulline (val-cit or vc) dipeptides linker reagents have with Flowering structure:
Wherein, Q is C1-C8Alkyl ,-O- (C1-C8Alkyl) ,-halogen ,-NO2Or-CN；And m is the integer of 0-4.
Can be had according to Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60 preparation Exemplary phe-lys (Mtr) dipeptides linker reagents of aminobenzyl group, and have a structure that
Wherein, Mtr is mono- 4- Methoxytrityl, and Q is C1-C8Alkyl ,-O- (C1-C8Alkyl) ,-halogen ,-NO2Or- CN；And m is the integer of 0-4 range.
" selfdecomposition connector " PAB (to amino benzyloxy carbamyl) connects drug moiety in antibody drug conjugate To antibody (Carl et al (1981) J.Med.Chem.24:479-480；Chakravarty et al(1983) J.Med.Chem.26:638-644；US 6214345；US20030130189；US20030096743；US6759509； US20040052793；US6218519；US6835807；US6268488；US20040018194；WO98/13059； US20040052793；US6677435；US5621002；US20040121940；WO2004/032828).In addition to PAB, divide certainly Other examples of the introns of solution include but is not limited to: (i) charge is similar to the aromatic compound such as 2- amino miaow of PAB group Azoles -5- carbinol derivatives (Hay et al. (1999) Bioorg.Med.Chem.Lett.9:2237), thiazole (US 7375078) PAB unit (de Groot et al (2001) J.Org.Chem.66:8815-8830) that is, more, extending；With it is o- Aminobenzene acetal or p-aminophenyl acetal；The styryl PAB analog (US 7223837) of (ii) confirmation.It can be used logical The introns of superamide key hydrolysis experience cyclisation, 4-Aminobutanoicacid amide (Rodrigues et al such as replace and unsubstituted (1995) Chemistry Biology 2:223), bicyclic [2.2.1] ring system and bicyclic [2.2.2] ring system that suitably replace (Storm et al (1972) J.Amer.Chem.Soc.94:5815) and 2- aminophenyl propionic acid (Amsberry, et al (1990)J.Org.Chem.55:5867).Eliminate (the Kingsbury et al (1984) containing drug amine replaced at glycine J.Med.Chem.27:1447 the example of useful selfdecomposition introns) and in the adc.
In one embodiment, valine-citrulline dipeptides PAB analog reagent has 2,6- 3,5-dimethylphenyl group And it has a structure that
There are the linker reagents for antibody drug conjugate of the invention to include but is not limited to: BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo group-EMCS, sulfo group-GMBS, Sulfo group-KMUS, sulfo group-MBS, sulfo group-SIAB, sulfo group-SMCC and sulfo group-SMPB and SVSB (succinimido-(4- ethylene Base sulfone) benzoic ether) and double-maleimide reagent: DTME, BMB, BMDB, BMH, BMOE, 1,8- be bis--maleimide Diethylene glycol (DEG) (BM (PEO)2) and 1,11- it is bis--maleimide triethylene glycol (BM (PEO)3), by Pierce Biotechnology, Inc., ThermoScientific, Rockford, IL and other reagent suppliers business can get.Double-maleimide reagent Allow that the free thiol group of the cysteine of antibody is connected to drug moiety, mark containing mercaptan in a manner of serially or simultaneously Note or connector intermediate.In addition to maleimide, reacted with the thiol group of antibody, PBD drug moiety or connector intermediate Other functional groups include iodo-acetamide, acetbromamide, vinylpyridine, disulphide, pyridyl disulfide, isocyanic acid Ester and isothiocyanates.
The other embodiments of linker reagents are: N- succinimido -4- (2- pyridylthio) valerate (SPP), N- succinimido -3- (2- pyridyl group two is thio) propionic ester (SPDP, Carlsson et al (1978) Biochem.J.173:723-737), succinimido -4- (N- Maleimidomethyl) hexamethylene -1- carboxylate (SMCC), iminothiolane (IT), the bifunctional derivative of polyurethane (such as dimethyl adipimide ester HCl), active ester be (such as Two succinimidyl suberates), aldehyde (such as glutaraldehyde), double azido compound (as bis- (to azidobenzoyls) oneself two Amine), double-diazo compound derivative (as bis- (to diazoniumbenzoyl)-ethylenediamines), diisocyanate (such as toluene 2,6- diisocyanate Ester) and double activated fluorine compounds (such as fluoro- 2,4- dinitrobenzene of 1,5- bis-).Pass through other commercial sources, such as Molecular Biosciences Inc. (Boulder, CO) also available useful linker reagents, or according to Toki et al (2002) J.Org.Chem.67:1866-1872；US 6214345；WO 02/088172；US 2003130189；US2003096743；WO 03/026577；WO 03/043583；Useful linker reagents can also be synthesized with step described in WO 04/032828.
Connector can be for being covalently attached to more than one drug moiety by branching, polyfunctional junction portion Dendritic type fittings (the US 2006/116422 of antibody；US2005/271615；de Groot et al(2003) Angew.Chem.Int.Ed.42:4490-4494；Amir et al(2003)Angew.Chem.Int.Ed.42:4494- 4499；Shamis et al(2004)J.Am.Chem.Soc.126:1726-1731；Sun et al(2002)Bioorganic& Medicinal Chemistry Letters 12:2213-2215；Sun et al(2003)Bioorganic&Medicinal Chemistry 11:1761-1768；King et al(2002)Tetrahedron Letters43:1987-1990).Branch Shaped polymer connector can increase the molar ratio of drug and antibody, that is, and drugloading rate is related to the effect of ADC.Therefore, when anti- When body only has a reactive cysteine thiol group, high amount of drug part can be linked by dendritic or branch Head connection.
One illustrative embodiments of dendritic type fittings have a structure that
Wherein, asterisk indicates the point for being connected to the position N10 of the part PBD.
Rc, end-capping group
The conjugate of the first aspect of the present invention can have end-capping group R at the position N10C.Compound E can have End-capping group RC。
In one embodiment, when conjugate is that wherein each monomer is the dimer of formula (A), a monomeric unit In group R10It is end-capping group RCOr group R10。
In one embodiment, when conjugate is that wherein each monomer is the dimer of formula (A), a monomeric unit In group R10It is end-capping group RC。
In one embodiment, when compound E is that wherein each monomer is the dimer of formula (E), a monomer list Group R in memberLIt is end-capping group RCOr the connector for being connected to antibody.
In one embodiment, when conjugate is that wherein each monomer is the dimer of formula (E), a monomeric unit In group RLIt is end-capping group RC。
Group R can be removed from the position N10 of the part PBDCTo residue N10-C11 imine linkage, carbinolamine, substituted methanol Amine, wherein QR11It is OSO3M, disulfide adducts, thiomethoxide amine, substituted thiomethoxide amine or substituted carbinolamine.
In one embodiment, RCRemovable blocking group be can be to residue N10-C11 imine linkage, methanol Amine, substituted carbinolamine or disulfide adducts, wherein QR11It is OSO3M.In one embodiment, RCIt is that can remove To the blocking group of residue N10-C11 imine linkage.
It is directed at and is used to remove group R10Required condition for example generates N10-C11 imine linkage, methanol under the same conditions Under conditions of amine etc., group R can be removedc.End-capping group plays the blocking group of the expection functional group at the position N10.Purport End-capping group not with antibody response.For example, RCWith RLIt is different.
In the synthesis of the dimer with imide monomers, the compound with end-capping group can be used as intermediate. Alternatively, the compound with end-capping group can be used as conjugate, wherein remove end-capping group from target location to produce Raw imines, carbinolamine, substituted carbinolamine etc..Therefore, in this embodiment, end-capping group is referred to alternatively as to remove in treatment Nitrogen-protecting group group, as defined in the earlier application WO 00/12507 of inventor.
In one embodiment, in cutting group R10Connector RLUnder conditions of, group R can be removedC.Therefore, one In a embodiment, end-capping group is cleavable under enzyme effect.
In an alternative embodiment, by connector RLIt is connected to before antibody, end-capping group can be removed.At this In embodiment, connector R is not being cutLUnder conditions of can remove end-capping group.
When compound includes functional group G1When forming the connection with antibody, in addition or unmasked G1Before, sealing end can be removed Group.
End-capping group can be used as to a part of blocking group strategy to ensure the only one monomeric unit in dimer It is connected to antibody.
End-capping group can be used as to the shelter (mask) of N10-C11 imine linkage.Imines can be needed in compound The time of functional group removes end-capping group.Carbinolamine, substituted carbinolamine and the curing that end-capping group is also described above The shelter of object adduct.
RCIt can be N10 blocking group, those groups as described in the earlier application WO 00/12507 in inventor. In one embodiment, RCIt is to treat upper removable nitrogen-protecting group group, such as in the earlier application WO 00/12507 of inventor Defined in.
In one embodiment, RCIt is carbamate protecting group.
In one embodiment, carbamate protecting group is selected from:
Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.
Optionally, carbamate protecting group is further selected from Moc.
In one embodiment, RCIt is the absence of the linker group R of the functional group for being connected to antibodyL。
Special consideration should be given to the R that those are carbamate by the applicationCGroup.
In one embodiment, RCIt is following group:
Wherein, asterisk indicates the point for being connected to the position N10, G2It is end-capping group, L3It is covalent bond or cleavable connector L1, L2It is covalent bond or is formed together selfdecomposition connector with OC (=O).
Work as L3And L2When being all covalent bond, G2Carbamate groups defined above are formed together with OC (=O).
As defined above, L1With R10It is related.
As defined above, L2With R10It is related.
A variety of end-capping groups are described below, including based on those of well known blocking group.
In one embodiment, L3It is cleavable connector L1, and L2Selfdecomposition connector is formed together with OC (=O). In this embodiment, G2It is Ac (acetyl group) or Moc or carbamate protecting group selected from the following:
Alloc, Fmoc, Boc, Troc, Teoc, Psec, Cbz and PNZ.
Optionally, carbamate protecting group is further selected from Moc.
In another embodiment, G2It is carboxyl groups-C (=O) G3, wherein G3Selected from alkyl (including naphthenic base, alkene Base and alkynyl), miscellaneous alkyl, heterocycle and aryl (including heteroaryl and carbon aryl (carboaryl)).These groups can be can Choose generation.When suitable, aryl group and L3Or L2Amino group can form amido bond together.When suitable, aryl group with L3Or L2Hydroxyl group can form ester bond together.
In one embodiment, G3It is miscellaneous alkyl.Miscellaneous alkyl group may include polyethylene glycol.Miscellaneous alkyl group can be with With the hetero atom for closing on aryl group, such as O or N, thus when suitable, and it is present in group L3Or L2In hetero atom formed Carbamate or carbonate group.
In one embodiment, G3Selected from NH2, NHR and NRR '.Preferably, G3It is NRR '.
In one embodiment, G2It is following group:
Wherein, asterisk expression is connected to L3Point, n is 0 to 6, and G4Selected from OH, OR, SH, SR, COOR, CONH2、 CONHR、CONRR’、NH2、NHR、NRR’、NO2And halogen.Group OH, SH, NH2It is shielded with NHR.In an embodiment party In formula, n is 1 to 6, and preferably n is 5.In one embodiment, G4It is OR, SR, COOR, CONH2、CONHR、CONRR’ And NRR '.In one embodiment, G4It is OR, SR and NRR '.Preferably, G4Selected from OR and NRR ', most preferably, G4It is OR. Most preferably, G4It is OMe.
In one embodiment, group G2It is:
Wherein, asterisk expression is connected to L3Point, and n and G4It is such as defined above.
In one embodiment, group G2It is:
Wherein, asterisk expression is connected to L3Point, n is 0 or 1, and m is 0 to 50, and G4Selected from OH, OR, SH, SR, COOR, CONH2、CONHR、CONRR’、NH2、NHR、NRR’、NO2And halogen.In one preferred embodiment, it is 0 that n, which is 1 and m, To 10,1 to 2, preferably 4 to 8 and most preferably 4 or 8.In another embodiment, it is 10 to 50 that n, which is 1 and m, excellent Selection of land is 20 to 40.Group OH, SH, NH2It is shielded with NHR.In one embodiment, G4Be OR, SR, COOR, CONH2, CONHR, CONRR ' and NRR '.In one embodiment, G4It is OR, SR and NRR '.Preferably, G4Selected from OR and NRR ', most preferably, G4It is OR.Preferably, G4It is OMe.
In one embodiment, group G2It is:
Wherein, asterisk expression is connected to L3Point and n, m and G4As defined above.
In one embodiment, group G2It is:
Wherein, n is 1-20, and m is 0-6, and G4Selected from OH, OR, SH, SR, COOR, CONH2、CONHR、CONRR’、NH2、 NHR、NRR’、NO2And halogen.In one embodiment, n is 1-10.In another embodiment, n is 10 to 50, preferably Ground is 20 to 40.In one embodiment, n is 1.In one embodiment, m is 1.Group OH, SH, NH2With NHR be by Protection.In one embodiment, G4It is OR, SR, COOR, CONH2, CONHR, CONRR ' and NRR '.In an embodiment In, G4It is OR, SR and NRR '.Preferably, G4Selected from OR and NRR ', most preferably, G4It is OR.Preferably, G4It is OMe.
In one embodiment, group G2It is:
Wherein, asterisk expression is connected to L3Point and n, m and G4As defined above.
In each of embodiment of above, G4It can be OH, SH, NH2And NHR.These groups are preferably protected 's.
In one embodiment, OH is protected with Bzl, TBDMS or TBDPS.
In one embodiment, SH is protected with Acm, Bzl, Bzl-OMe, Bzl-Me or Trt.
In one embodiment, NH is protected with Boc, Moc, Z-Cl, Fmoc, Z or Alloc2Or NHR.
In one embodiment, group G2With group L3Combination exists, group L3It is dipeptides.
End-capping group, which is intended to be not used in, is connected to antibody.Accordingly, there exist the other monomers in dimer to be used as via connecing Head is connected to the point of antibody.It is therefore preferable that the functional group being present in end-capping group is not useable for and antibody response.Cause This, is preferably avoided reactive functional groups, such as OH, SH, NH2,COOH.However, if protected, as described above, this Kind functional group can reside in end-capping group.
Embodiment
Embodiments of the present invention include ConjA, wherein antibody is as defined above.
Embodiments of the present invention include ConjB, wherein antibody is as defined above.
Embodiments of the present invention include ConjC, wherein antibody is as defined above.
Embodiments of the present invention include ConjD, wherein antibody is as defined above.
Embodiments of the present invention include ConjE, wherein antibody is as defined above.
As described above, some embodiments of the present invention do not include ConjA, ConjB, ConjC, ConjD and ConjE.
Drugloading rate
Drugloading rate is the average number of every antibody (for example, antibody) PBD drug.When the compound of the present invention is bonded to half Guang When propylhomoserin, drugloading rate can be in 1 to 8 drug (D of every antibodyL) in the range of, that is, wherein 1,2,3,4,5,6,7 and 8 drug Some covalent is connected to antibody.The composition of conjugate includes the set for being combined with the antibody of drug within the scope of 1 to 8.When this When the compound of invention is bonded to lysine, drugloading rate can be in 1 to 80 drug (D of every antibodyL) in the range of, although 40, 20,10 or 8 upper limits may be preferred.The composition of conjugate includes being combined with 1 to 80,1 to 40,1 to 20,1 to 10 Or within the scope of 1 to 8 the antibody of drug set.
Prepared in product in the ADC from association reaction, the average number of every antibody drug can by usual manner, as UV, Reverse hplc, HIC, mass spectrum, ELISA measurement and electrophoresis characterization.It can also determine with the quantitative distribution of the p ADC indicated.Pass through ELISA can determine average value (Hamblett et al (2004) Clin.Cancer for preparing p in product in specific ADC Res.10:7063-7070；Sanderson et al(2005)Clin.Cancer Res.11:843-852).However, by anti- Body-antigen binding and the detection of ELISA limit, the distribution of p (drug) value is unrecognizable.In addition, for detecting antibody-drug The ELISA measurement of conjugate not can determine where drug moiety is being connected to antibody, such as heavy chain or light chain segments or specific Amino acid residue.In some cases, it can realize that (wherein p is homogeneous ADC by way of such as reversed-phase HPLC or electrophoresis Specific value) separation, purifying and characterization with the ADC with other drugloading rates.This technology is also applied for other kinds of knot Close object.
For certain antibody-drug conjugates, p can be limited by the number of connection site on antibody.For example, antibody can be with With only one or there are multiple cysteine thiol groups, or can have only one or there are multiple sufficiently reactive sulphur Alcohol groups can connect connector by the thiol group.Higher drugloading rate, such as p > 5 can cause certain antibody-drugs The aggregation of conjugate, insoluble, toxicity or cell permeability are lost.
In general, the drug moiety less than theoretical maximum amount is bound to antibody by association reaction.For example, antibody can wrap The lysine residue not reacted with agent-linker intermediate (D-L) or linker reagents containing many.Only most reactive lysine Group can be reacted with amine reactivity linker reagents.In addition, only most reactive cysteine residues can be with thiol reaction Property linker reagents reaction.In general, antibody does not include cysteine thiol that many (if any) is dissociated and reactive Group, can connect to drug moiety.Most of cysteine mercaptan residues in the antibody of compound exist as disulphide bridges, And it must be restored under part or all of reducing condition with reducing agent such as dithiothreitol (DTT) (DTT) or TCEP.It can be with The drugloading rate (drug/antibody ratio) of various ways control ADC, comprising: (i) limits agent-linker intermediate (D-L) or connector examination Molar excess of the agent relative to antibody, (ii) limit association reaction time or temperature, and (iii) is repaired for cysteine mercaptan The part of decorations or restricted reducing condition.
Certain antibody have disulphide in reducible chain, that is, cysteine bridge.For and linker reagents combination, Antibody can be made to become that there is reactivity by being handled with reducing agent such as DTT (dithiothreitol (DTT)).Therefore, theoretically each half Guang Propylhomoserin bridge will form two reactive nucleophilic thiol bodies.By obtaining the lysine and 2- imino group thiophene of conversion of the amine as mercaptan The reaction of pheno (Traut ' s reagent), other nucleophilic group can be introduced in antibody.Pass through design 1,2,3,4 or more Cysteine residues (for example, the mutant antibodies of preparation comprising one or more non-natural cysteine residues) can be by mercapto Group is introduced to antibody (or their segment).The introduction of US 7521541 is anti-by introducing reactive cysteine amino acids design Body.
Cysteine residues can be designed at the reaction site in antibody and it is not formed in chain or intramolecular disulfide Key (Junutula, et al., 2008b Nature Biotech., 26 (8): 925-932；Dornan et al(2009) Blood 114(13):2721-2729；US 7521541；US 7723485；WO2009/052249).The hcy thiolactone of design Alcohol can be with linker reagents with thiol reactivity, electrophilic group (such as maleimide or alpha-halogenate amide) or of the invention The reaction of agent-linker reagent is to form the antibody of the cysteine with design and the ADC of PBD drug moiety.Therefore, Ke Yishe The position of meter, control and known drug part.Due to design cysteine thiol group usually with high yield it is anti-with mercaptan Answering property linker reagents or the reaction of agent-linker reagent, it is all to can control drugloading rate.Pass through the single site in heavy chain or light chain Displacement is to design IgG antibody to introduce cysteine amino acids and generate two new cysteines on symmetrical antibody.Pass through knot Close product ADC close to homogeney, the drugloading rate close to 2 may be implemented.
Alternatively, as Axup et al. ((2012), Proc Natl Acad Sci U S A.109 (40): 16101- 16116) described, by the way that antibody design to include unnatural amino acid in their heavy chain and/or light chain, may be implemented Locus specificity combines.Unnatural amino acid provides other advantage, it can designs orthogonal chemistry and carrys out jointing reagent And drug.
When the more than one nucleophilic or electrophilic group of antibody and connecing before agent-linker intermediate or drug moiety reagent When head reagent reaction, then obtained product is the distribution with the drug moiety (for example, 1,2,3 etc.) for being connected to antibody The mixture of ADC compound.Liquid chromatography, as polymer reverse phase (PLRP) and hydrophobic interaction can pass through drugloading rate value Compound in separating mixture.The ADC with single drugloading rate value (p) can be separated prepares product, however, these single loads Dose value ADC still can be a variety of mixtures, because drug moiety can be connected to the different loci of antibody via connector On.
Therefore, antibody-drug combining compositions of the invention include the mixture of antibody-drug binding compounds, wherein Antibody has one or more PBD drug moiety, and wherein, drug moiety can be connected to anti-at different aminoacids residue Body.
In one embodiment, the dimer Pyrrolobenzodiazepines of every antibodyThe average number of group 1 to In the range of 20.In some embodiments, range is selected from 1 to 8,2 to 8,2 to 6,2 to 4 and 4 to 8.
In some embodiments, there are a dimer Pyrrolobenzodiazepines in every antibodyIncluding other shapes Formula
It unless otherwise prescribed, hereinbefore include well-known ion, salt, solvate and the protection of these substituent groups Form.For example, referring to that carboxylic acid (- COOH) further includes its anion (carboxylate radical) form (- COO-), salt or solvate, and Conventional protected forms.Similarly, refer to that amino includes the protonated form (- N of amino+HR1R2), salt or solvate, for example, The conventional protected forms of hydrochloride and amino.Similarly, refer to that hydroxyl further includes its anionic form (- O-), salt or solvent Compound and conventional protected forms.
Salt
It is can be convenient or it is expected that preparation, purifying and/or processing reactive compound corresponding salt, for example, pharmacy Upper acceptable salt.The example of pharmaceutically acceptable salt is discussed at Berge, et al., J.Pharm.Sci., 66,1-19 (1977)。
For example, if compound is anionic compound, or can be the functional group of anion (for example,-COOH with it It can be-COO-), then can be with suitable salt forming cation.The example of suitable inorganic cation includes but is not limited to Alkali metal ion such as Na+And K+, alkaline earth metal cation such as Ca2+And Mg2+And other cations such as Al+3.Suitable organic sun The example of ion includes but is not limited to ammonium ion (i.e. NH4 +) and ammonium ion (such as the NH that replaces3R+、NH2R2 +、NHR3 +、NR4 +)。 The example of some suitable substituted ammonium ions is the ammonium ion that those replace, and is originated from: ethamine, diethylamine, dicyclohexyl amine, three Ethamine, butylamine, ethylenediamine, ethanol amine, diethanol amine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine (tromethamine) and amino acid, such as lysine and arginine.The example of common quaternary ammonium ion is N (CH3)4 +。
If compound is cationic compound, or can be the functional group (such as-NH of cation with it2Can be- NH3 +), then can be with suitable anion forming salt.The example of suitable inorganic anion include but is not limited to be originated from it is following Those of inorganic acid: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid.
The example of suitable organic anion includes but is not limited to be originated from those of following organic acid: 2- acetyloxy phenyl first Acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediamine tetra-acetic acid, two sulphur of ethane Acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyl second sulphur Acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, glactaric acid, oleic acid, oxalic acid, palmitinic acid, flutters acid, pantothenic acid, benzene at lactic acid Acetic acid, benzene sulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzene sulfonic acid, tartaric acid, toluenesulfonic acid, three Fluoroacetic acid and valeric acid.The example of suitable macromolecule organic anion includes but is not limited to those macromolecule organic anions, From following polymeric acid: tannic acid, carboxymethyl cellulose.
Solvate
Coordinative solvent compound that is can be convenient or desirably preparing, purify and/or handle reactive compound.Term " solvate " is come with conventional sense using being used to refer to solute (such as salt of reactive compound, reactive compound) herein With the compound of solvent.If solvent is water, solvate is referred to as hydrate with can be convenient, for example, monohydrate, two hydrations Object, trihydrate etc..
The present invention includes that the compound of solvent is wherein added across the imine linkage of the part (across) PBD, is shown hereafter, Wherein solvent is water or alcohol (RAOH, wherein RAIt is C1-4Alkyl):
The carbinolamine and methanol amidogen ether form that these forms can be referred to as PBD are (such as above with respect to R10Part in institute Description).The balance of these equatioies depends on the condition of discovery compound and the characteristic of part itself.
For example, these specific compounds can be separated in solid form by freeze-drying.
Isomers
Certain compounds of the invention can with one or more specific geometric formats, optical form, enantiomeric form, Diastereomer form, epimeric form, dystopy isomers (atropic) form, stereoisomer form, tautomer Form, conformational forms or different head isomers (anomeric) form, including but not limited to cis and trans；E type and Z-type；C formula, t Formula and r formula；Interior form and outer form；R type, S type and meso-form；D type and L-type；D type and l type；(+) and (-) form；Keto-acid, Enol form and enolate-forms；Cis and trans；To oblique formula and anticline form；Alpha form and beta form；Axial form peace Volt form；Boat form, turns round formula, envelope type and half-chair at chair form；And their combination, hereinafter collectively referred to as " isomers " (or " isomeric forms ").
Term " chirality " refers to non-overlap (non-overlapping, non-superimposability) property with mirror image companion The molecule of matter, and term " achirality " refers to it is the molecule being overlapped on their mirror image companion.
Term " stereoisomer " refer to identical chemical component, but spatially atom or group arrange it is different Compound.
" diastereoisomer " refers to two or more chiral centres and its molecule is not mutually another mirror The stereoisomer of picture.Diastereoisomer has different physical properties, for example, fusing point, boiling point, spectral property and reaction Property.It can be under high redissolution analytical procedure such as the mixture of electrophoresis and chromatographic isolation diastereoisomer.
" enantiomter " refers to two kinds of stereoisomers of compound, is mutual non-superimposable mirror images.
Spatial chemistry used herein limits and regulation usually follows S.P.Parker, Ed., McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company,New York；and Eliel, E.and Wilen, S., " Stereochemistry of Organic Compounds ", John Wiley&Sons, Inc.,New York,1994.The compound of the present invention may include asymmetric or chiral centre, therefore different with different solids Structure body form exists.Be intended to the compound of the present invention all stereoisomer forms include but is not limited to diastereoisomer, Enantiomter and atropisomer (atropisomer) and their mixture (such as racemic mixture), form this hair Bright a part.Many organic compounds exist with optical active forms, that is, they have the Plane Rotation for making linearly polarized light Ability.In description optically active compound, prefix D and L or R and S be used to indicate molecule about it chiral centre it is exhausted To configuration.The symbol rotated using the linearly polarized light of prefix d and l or (+) and (-) Lai Zhiding chemicals, wherein (-) or l are Refer to that compound is left-handed.Prefix is (+) or the compound of d is dextrorotation.For given chemical structure, these alloisomerisms Body is identical other than they are mutual mirror images.Specific stereoisomer can be by referred to as enantiomter, and passes through The mixture of this isomers is often known as enantiomeric mixture.The 50:50 mixture of enantiomter is known as racemic Mixture or racemic modification, this can occur in the chemical reaction for not having stereoselectivity or stereospecificity or in the process. Term " racemic mixture " and " racemic modification " refer to that two kinds of equimolars without optically active enantiomter substance are mixed Close object.
It should be noted that unless as follows be directed to what tautomeric forms were discussed, from term " isomers " (such as at this It is as used herein) structure (or construction) isomers is clearly excluded (that is, its difference is connection from atom to atom and not only exists Isomers in the position of atom in space).For example, referring to methoxyl group (- OCH3) be not interpreted to refer to its structure Isomers (methylol ,-CH2OH).Similarly, refer to that Chloro-O-Phenyl is not interpreted to refer to its constitutional isomer (m-chloro Phenyl).However, referring to that a class formation may include structural isomer forms (such as the C within the scope of falling in the above-mentioned type1-7 Alkyl includes n-propyl and isopropyl；Butyl includes normal-butyl, isobutyl group, sec-butyl and tert-butyl；Methoxyphenyl includes neighbour Methoxyphenyl, m-methoxyphenyl and p-methoxyphenyl).
Above-mentioned exclusion is not related to tautomeric forms, such as exists, for example, following tautomer centering: ketone/enol (as shown below), imines/enamine, amide/imino group alcohol, amidine/amidine, nitroso/oxime, thioketones/alkene mercaptan, N- nitroso/hydroxyl It is in base azo and nitro/isonitro for example, keto-acid, enol form and enolate-forms.
Term " tautomer " or " tautomeric forms " refer to the constitutional isomer of different-energy, pass through low energy It is interconvertible for measuring potential barrier.For example, proton tautomer (also referred to as protic tautomer) includes moving by proton The enantiotropy of shifting, such as keto-enol and imine-enamine isomerizations.Valence tautomers include passing through some bonding electrons Recombination (reorganization) enantiotropy.
It should be noted that term " isomers " particularly including the compound that there are one or more isotopes to replace.For example, H can be any isotope form, including1H、2H (D) and3H(T)；C can be any isotope form, including12C、13C and14C；O can be any isotope form, including16O and18O；Etc..
The example for the isotope that can be bound in the compound of the present invention includes the same of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine Position element, such as, but not limited to,2H (deuterium, D),3H (tritium),11C、13C、14C、15N、18F、31P、32P、35S、36Cl and125I.Of the invention is each The compound of kind isotope labelling, such as radioactive isotope such as 3H, 13C, 14C are incorporated into these compounds.It is such same The compound of position element label can be used for being metabolized research, Reaction kinetics research, detection or imaging technique, as positive electron tomography is swept Imaging (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distributional analysis are retouched, or In the radiation treatment of patient.Deuterium-labeled or substituted therapeutic compound of the invention can have improved DMPK (drug metabolism and pharmacokinetics) property is related to distribution, metabolism and excretion (ADME).It is taken with heavier isotope (such as deuterium) In generation, can provide certain treatment advantages generated by higher metabolic stability, for example, Half-life in vivo increase or required Dosage is reduced.The compound of 18F label is useful to PET or SPECT research.The compound of isotope labelling of the invention is logical Following preparation can often be passed through: implementing the step of disclosing in reaction scheme or embodiment as described below and preparation example (journey Sequence), isotope-labeled reagent is replaced with the reagent for the isotope labelling being easy to get.In addition, with heavier isotope, especially It is deuterium (that is, 2H or D) substitution, certain treatment advantages generated by higher metabolic stability can be provided, for example, in vivo Half-life period increases or required dosage reduces or therapeutic index increases.It should be understood that the deuterium in context is regarded as replacing Base.The concentration of this higher isotope (especially deuterium) can be limited by isotope enrichment factor.In chemical combination of the invention It is any not clearly indicate any stable isotope for referring to the atom for the atom of specific isotope in object.
Unless otherwise stated, the particular compound otherwise mentioned include all these isomeric forms, including its (all or Partly) racemic and other mixtures.Preparation (for example, asymmetric syntheses) and separation (example for this isomeric forms Such as, fractional crystallization and chromatography) method be known in the art, or adjust side teaching herein by known methods Method or known method are readily available.
Bioactivity
In vitro cell proliferation assay
In general, passing through the cytotoxicity or cell inhibitory activity of following measurement antibody-drug conjugates (ADC): will have The mammalian cell of receptor protein is exposed to the antibody of ADC in cell culture medium；It cultivates one section of cell about 6 hours to about 5 days Period；And measurement cell viability.External test based on cell is used to measure the survival ability of ADC of the invention The induction (caspase activation) of (proliferation), cytotoxicity and cell death.
The vitro efficacy of antibody-drug conjugates can be measured by cell proliferating determining. Luminescent cell viability analyzer is commercially available (Promega Corp., Madison, WI), is based on Homogeneity measuring method (the US patent No. 5583024 of the recombinant expression of Coleoptera luciferase；5674713 Hes 5700670).For the cell proliferating determining based on there are the number for quantitatively determining Survival Cells in culture medium of ATP, which is generation Thank to instruction (Crouch et al (1993) J.Immunol.Meth.160:81-88 of competent cell；US 6602677).96 It is carried out in orifice plateMeasurement makes it that can be subjected to inspection (the Cree et al of high throughput screening (HTS) (1995)AntiCancer Drugs 6:398-404).Determination step of the same race be related to directly adding single agents (Reagent) to culture supplement serum culture medium cell.Cell washing removes culture medium and repeatedly moves Liquid step is unwanted.Add reagent and mixing after, in 10 minutes system detect in 384 orifice plates down to 15 cells/ Hole.ADC continuous processing cell can be used, or can handle cell and separate it with ADC.In general, simple process is (that is, 3 is small When) cell effect identical as the cell of continuous processing is shown.
" addition-mixing-measurement " mode of homogeneity causes cell lysis to act on and proportional to the amount there are ATP shine (luminescent) generation of signal.The amount of ATP and the number for the cell being present in culture medium are directly proportional.Measurement generates " growth type " luminous signal generated by luciferase reaction, depending on the cell class used Type and culture medium have the half-life period for being typically larger than 5 hours.Survival Cells react on relative luminescent units (RLU).Pass through Recombinate the adjoint conversion of firefly luciferase and ATP to AMP and the generation of photon, substrate B eetle Luciferin (beetle Fluorescein) it is oxidized decarboxylation.
The vitro efficacy of antibody-drug conjugates can also be measured by cytotoxicity assay.It is washed with PBS, with pancreas egg The adherent cell that white enzyme is separately cultured is diluted in the complete medium comprising 10%FCS, be centrifuged, be resuspended in it is fresh In culture medium, and counted with hemacytometer (haemocytometer).Directly count suspension culture.Suitable for counting Monodisperse cell suspension may be needed by repeating to supply gas to stir suspended matter to break up cell mass.
Cell suspension is diluted to desired inoculum density and is dispersed in 96 orifice plates of black (100 hole μ l/). The plate of adherent cell system is incubated for overnight to allow to adhere to.The suspension cell of culture can be used on the day of inoculation.
The stock solution (1ml) of ADC (20 μ g/ml) is prepared in cell culture medium appropriate.By the way that sequentially 100 μ l are turned The cell culture medium for moving to 900 μ l, a series of 10 times of dilutions of preparation deposit ADC in 15ml centrifuge tube.
Before with cell suspending liquid (100 μ l) bed board, four repeating holes of each ADC dilution (100 μ l) are dispersed In 96 hole blackboards, 200 μ l of final volume is obtained.Control wells receive cell culture medium (100 μ l).
If the doubling time of cell line is greater than 30 hours, ADC is incubated for 5 days, otherwise completes to be incubated in four days.
In incubation time section finally, with the blue measurement assessment cell viability of alma (Alamar).By alma indigo plant (Invitrogen) it is dispersed on entire plate (20 hole μ l/) and is incubated for 4 hours.In the quick plate reader of Varioskan with 570nm excitation, 585nm emission measurement alma indigo plant fluorescence.Compared with the mean fluorecence in control wells, in the hole by ADC processing Mean fluorecence calculate percentage cell survival rate.
Purposes
Conjugate of the invention can be used for providing PBD compound in target location.
Target location is preferably the cell colony being proliferated.Antibody is the antigen for being present on proliferating cell population Antibody.
In one embodiment, with the amount for the antigen being present in proliferating cell population (for example, tumor cell colonies) It compares, antigen is not present or is present in non-proliferative cell colony with reduction level.
In target location, connector can be cut, to discharge compound R elA, RelB, RelC, RelD or RelE.Therefore, Conjugate can be used to compound R elA, RelB, RelC, RelD or RelE being selectively provided to target location.
Connector can be cut by the digestion for being present in target location.
Target location can be external, internal or external.
Antibody-drug conjugates (ADC) compound of the invention includes having for those of anticancer activity effectiveness.Specifically Ground, compound include combining, that is, the antibody of PBD drug moiety (that is, toxin) is covalently attached to by connector.When drug does not have When being bound to antibody, PBD drug has cytotoxic effect.Therefore, by and the combination of antibody adjust PBD drug moiety Bioactivity.The cytotoxic reagent of effective dose is selectively delivered to tumour by antibody-drug conjugates (ADC) of the invention Tissue, so as to realize higher selectivity, that is, less effective dose.
Therefore, on the one hand, the present invention provides the combination compounds for being described herein for using in the treatment.
Further, it also provides and is described herein for the conjugate used in the treatment of proliferative disease Compound.The second aspect of the present invention, which provides, combines compounds preparing the use in the drug for treating proliferative disease On the way.
Those of ordinary skill in the art can be readily determined whether candidate conjugate treats any particular cell types Proliferative disorders.For example, describing such measuring method in the following examples, they may be conveniently used assessment by specific Activity provided by compound.
Term " proliferative disease " is related to the unwanted or uncontrolled cell of undesirable over or abnormal cell Hyperplasia, e.g., angiogenic or Hyperplastic growth (either in vitro or in vivo).
The example of proliferative disorders is including but not limited to benign, deteriorates preceding and malignant cell proliferation, including but not limited to newly Biology and tumour (for example, histocytoma, glioma, astrocytoma, osteoma), cancer (such as lung cancer, Small Cell Lung Cancer, Human primary gastrointestinal cancers, intestinal cancer, colon cancer, breast cancer, oophoroma, prostate cancer, carcinoma of testis, liver cancer, kidney, bladder cancer, cancer of pancreas, the cancer of the brain, Sarcoma, osteosarcoma, Kaposi sarcoma, melanoma), lymthoma, leukaemia, psoriasis, osteopathy, fibroproliferative disease (such as The fibroproliferative disease of connective tissue) and atherosclerosis.Cancer of special interest include but is not limited to leukaemia and Oophoroma.
It can treat any kind of cell, including but not limited to lung, gastrointestinal tract (including for example, intestines, colon), cream (cream Room), ovary, prostate, liver (liver), kidney (kidney), bladder, pancreas, brain and skin.
Cancer of special interest includes but is not limited to prostate cancer.
And have been shown that PSMA is significantly expressed in the new vessels of non-prostate solid tumor, the non-prostate entity Tumor includes colon tumor, lacteal tumor, bladder tumor, Vipoma, nephroncus and melanoma, but does not include normal hemangioma.Therefore, PSMA Specific ADC can be used for the non-tumor of prostate that treatment has PSMA positive new vessels.
It is expected that antibody-drug conjugates (ADC) of the invention can be used for treating a variety of diseases or disorder, for example, by swelling The overexpression of tumor antigen characterizes.Exemplary conditions or hyperplasia sexual disorder include benign or malignant tumour；Leukaemia, blood and Lymphoid malignancies.Other include disorder below: neuron, Deiter's cells, astrocyte, hypothalamus , body of gland, macrophage, epithelium, matrix, blastocoele, inflammatory, blood vessel generates and immunologic, packet Include autoimmune disorders.
In general, disease to be treated or disorder are hyperproliferative diseases, such as cancer.The reality of cancer to be treated herein Example includes but is not limited to cancer, lymthoma, blastoma, sarcoma and leukaemia or lymphoid malignancy.This cancer it is more specific Ground example include squamous cell carcinoma (for example, epithelial squamous cell cancer), lung cancer (including Small Cell Lung Cancer, non-small cell lung cancer), The gland cancer of the squamous cell carcinoma of lung and lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including human primary gastrointestinal cancers), cancer of pancreas, plastic Cell plastid tumor, cervical carcinoma, oophoroma, liver cancer, bladder cancer, hepatoma, breast cancer, colon and rectum carcinoma, colorectal cancer, son Endometrial carcinoma or uterine cancer, salivary-gland carcinoma, kidney or renal cancer, prostate cancer, carcinoma of vulva, thyroid cancer, liver tumour, anus Cancer, carcinoma of penis and head and neck cancer.
Can be used in the treatment ADC autoimmune disease include rheumatic disease (as example, rheumatoid arthritis, Sjogren syndrome, chorionitis, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody Syndrome and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorder are (as example, inflammatory bowel disease (example Such as, ulcerative colitis and Crohn disease), autoimmune gastritis and pernicious anaemia, oneself immunity hepatitis, primary biliary Property cirrhosis, primary sclerotic cholangitis and chylous diarrhea, vasculitis is (as example, the relevant vasculitis of ANCA, including Qiu-are applied Vasculitis, Wegner's granulomatosis and panarteritis), Autoimmune neuropathies disorder is (e.g., for example, multiple sclerosis, slanting eye Myoclonic syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease and autoimmune are more Send out nerve lesion), kidney trouble (as example, glomerulonephritis, Goodpasture's syndrome and Bei Geershi disease), itself Immune dermatosis disorder is (as example, psoriasis, nettle rash, morbilli, pemphigus vulgaris, bullous pemphigoid and skin are red Yabbi sore), hematology disorder is (as example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura And autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune auditory disorder is (as example, inner ear Disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant and autoimmune endocrine disturbance (as example, Diabetes associated autoimmune disease such as insulin-dependent diabetes mellitus (IDDM), Addison's disease and Autoimmune Thyroid Sick (for example, Graves disease and thyroiditis)).Preferred this disease includes, for example, rheumatic arthritis, exedens It is the relevant vasculitis of colitis, ANCA, lupus, multiple sclerosis, Sjogren syndrome, Graves disease, IDDM, pernicious poor Blood, thyroiditis and glomerulonephritis.
Treatment method
Conjugate of the invention can use in treatment method.There is also provided a kind of method for the treatment of, including to needing The subject wanted gives the combination compounds of the invention of therapeutically effective amount.Term " therapeutically effective amount " is to be enough to show to trouble The amount that person benefits.Above-mentioned benefit, which can be, at least improves a kind of symptom.The actual amount given and the velocity and time given Process will depend on property to be treated and seriousness.Treatment prescription, such as dosage determine, are in omni-doctor and other doctors The Limitation on Liability in.
It can individually give or be combined with other treatment and give the compound of the present invention, while give or sequence is to prefetching Certainly in illness to be treated.The example for the treatment of and therapy includes but is not limited to that chemotherapy (gives activating agent, including such as medicine Object, such as chemotherapy)；Operation；And radiotherapy.
Do not consider mechanism of action, " chemotherapeutics " is chemical compound useful in cancer treatment.The classification packet of chemotherapeutics Include but be not limited to Alkylators, antimetabolite, spindle poisonous plant alkaloid, cytotoxicity/antitumor antibiotics, topoisomerase Enzyme inhibitor, antibody, photosensitizer and kinase inhibitor.Chemotherapeutics is included in chemical combination used in " targeted therapies " and conventional chemotherapy Object.
The example of chemotherapeutics include: Tarceva (Genentech/OSI Pharm.), more west he Match (Sanofi-Aventis), 5-FU (fluorouracil, 5 FU 5 fluorouracil, CAS No.51-21-8), Ji His shore of west (Lilly), PD-0325901 (CAS No.391210-10-9, Pfizer), cis-platinum (cis- two Amine, dichloro platinum (II), CAS No.15663-27-1), carboplatin (CAS No.41575-94-4), taxol ( Bristol-Myers Squibb Oncology, Princeton, N.J.), Herceptin ( Genentech), Temozolomide (bicyclic [4.3.0] the nonyl- 2,7,9- triolefin -9- of 4- methyl -5- oxo -2,3,4,6,8- pentaaza Formamide, CAS No.85622-93-1,Schering Plough), he not Former times sweet smell ((Z) -2- [4- (1,2- diphenyl but-1-ene base) phenoxy group]-N, N- dimethyl amine,) and Doxorubicin Akti-1/2, HPPD and rapamycin.
More examples of chemotherapeutics include: oxaliplatinSanofi), bortezomib (Millennium Pharm.), sotan (SU11248, Pfizer), Letrozole (Novartis), imatinib mesylate (Novartis), (Mek inhibits XL-518 Agent, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (AstraZeneca), Calciumlevofolinate (folinic acid), rapamycin (sirolimus,Wyeth), Lapatinib (GSK572016, Glaxo Smith Kline), Lip river that Method Buddhist nun (SARASARTM, SCH 66336, Schering Plough), Sorafenib (BAY43-9006, Bayer Labs), Gefitinib (AstraZeneca), Irinotecan (CPT- 11, Pfizer) pyrrole method Buddhist nun (ZARNESTRA, is replacedTM, Johnson&Johnson), ABRAXANETM(be free of polyoxyethylene caster Oil), the albumin nano of taxol design preparation (American Pharmaceutical Partners, Schaumberg, Il), Vande Thani (rINN, ZD6474,AstraZeneca), Chlorambucil, AG1478, AG1571 (SU5271；Sugen), sirolimus (Wyeth), pazopanib (GlaxoSmithKline), camphane Buddhist (Telik), phosphinothioylidynetrisaziridine and cyclophosphamideAlkyl sulfonic ester is such as Busulfan, Improsulfan and piposulfan；Aziridines such as Benzodepa (benzodopa), carboquone, Meturedepa (meturedopa) and urethimine (uredopa)；Ethylenimines and methylmelamines (methylamelamines Including hemel, triethylenemelamine, triethylphosphoramide, triethylene thiophosphamide and three (methylamelamines), Methylol melamine (trimethylolomelamine)；Lactone (especially its pungent and its octanone of Bradley of Bradley)；Camptothecine (including The class topotecan of synthesis)；Bryostatin；callystatin；CC-1065 (including it] Adozelesin, Carzelesin and ratio It rolls over and carrys out new synthetic analogues)；Cryptophycin (the especially cryptophycin 8 of 1 sum of cryptophycin)；Dolastatin；Times carcinomycin (including close At analog, KW-2189 and CB1-TM1)；Eleutherobin (soft coral alcohol)；Water ghost any of several broadleaf plants alkali；Sarcodictyin (branch of crawling Coral alcohol)；spongistatin；Mustargen for example Chlorambucil, Chlornaphazine, chlorine phosphamide (chlorophosphamide), it is female not Take charge of spit of fland, ifosfamide, mechlorethamine, mechlorethamine oxide salt acidulants, melphalan, novembichin, Phenesterin, pennisetum mustard, trofosfamide, uracil mastard；Nitroso ureas such as Carmustine, chloramphenicol, good fortune are not Take charge of spit of fland, lomustine, Nimustine and ranimnustine；Antibiotic such as enediyne antibiotic (such as in Calicheamicin, card Miramycin γ 1I, Calicheamicin Ω I1 (Angew Chem.Intl.Ed.Engl. (1994) 33:183-186)；Up to endomycin (dynemicin), endomycin A is reached；Diphosphonate, such as clodronate；Ai Sipeila mycin；And neoearcinostain chromophore and Related chromoprotein enediyne antibiotic chromophore), aclacinomycin, D actinomycin D, authramycin, azaserine, it is rich come It is mycin, D actinomycin D, carabicin, carminomycin, carzinophillin, chromomycin (chromomycinis), dactinomycin D, soft red Mycin, Detorubicin, 6- diazonium -5- oxn-l-norieucin, morpholino-adriamycin, cyanomorpholino-doxorubicin, 2- pyrroles Quinoline base-adriamycin and deoxy doxorubicin, epirubicin, esorubicin, idarubicin, Nemorubicin, marcellomycin, mitogen Mycin for example mitomycin C, mycophenolic acid, nogalamycin, olivomycin class, Peplomycin, porfiromycin, puromycin, three-iron Ah Mycin, rodorubicin, streptonigrin, streptozotocin, tuberculin (tubercidin), ubenimex, Zinostatin, the soft ratio of assistant Star；Antimetabolite such as methotrexate (MTX) and 5 FU 5 fluorouracil (5-FU)；Folacin for example denopterin, methotrexate (MTX), pteropterin, Trimetrexate；Purine analogue such as fludarabine, Ismipur, thiapurine, thioguanine；Pyrimidine analogue such as Anxi he Shore, azacitidine, 6- azauridine, Carmofur, cytarabine, di-deoxyuridine, doxifluridine, enocitabine, floxuridine；It is male Hormone such as Calusterone, Masterone, epithioandrostanol, Mepitiostane, Testolactone；Anti- adrenal gland such as aminoglutethimide (aminoglutethimide), mitotane, Trilostane；Folic acid supplement such as folinic acid；Aceglatone (aceglatone)；Aldophosphamideglycoside；Amino-laevulic acid；Eniluracil；Amsacrine；bestrabucil；Bisantrene；According to Up to Qu Sha (edatraxate)；Defosfamide (defosfamide)；Demecolcine (Demecolcine)；Diaziquone； elfornithine；Elliptinium Acetate；Epothilones；Ethoglucid；Gallium nitrate；Hydroxycarbamide；Lentinan；Lonidamine (lonidainine)；Maytansinol (maytansinoid) such as maytansine (maytansine) and ansamitocin (ansamitocin)；Mitoguazone；Mitoxantrone；mopidanmol；Nitrazine (nitragin (nitraerine))；Spray department he Fourth；Benzene carrys out beautiful spy；Pirarubicin；Losoxantrone；Podophyllic acid (podophyllinic acid)；2- ethyl hydrazine；Procarbazine；Polysaccharide compound (JHS Natural Products, Eugene, OR)；Razoxane；Rhizomycin；Sizofiran；Spirogermanium (spirogermanium)；Tenuazonic acid；Triethyleneiminobenzoquinone；2,2 ', 2 "-trichlorotriethylamines；Trichothecenes toxin Class (especially T-2 toxin, myconomycin A (verracurin A), Roridine A and anguidin)；Urethane；Long fields for spring sowing It is pungent；Dacarbazine；Mannomustin；Dibromannitol；Mitolactol；Pipobroman；gacytosine；Cytarabine ("Ara-C")；Cyclophosphamide；Phosphinothioylidynetrisaziridine；6- thioguanine；Purinethol；Methotrexate (MTX)；Platinum analogs such as cis-platinum and carboplatin； Vinblastine；Etoposide (VP-16)；Ifosfamide；Mitoxantrone；Vincristine；VinorelbineNovantrone (novantrone)；Teniposide；Edatrexate；Daunorubicin；Aminopterin；Card training His shoreRoche)；Ibandronate；CPT-11；Topoisomerase enzyme inhibitor RFS2000；Difluoromethyl bird ammonia Sour (DFMO)；Retinoids class (biostearin) such as retinoic acid；Pharmaceutically acceptable salt, acid and the derivative of any of the above Object.
Further include in the restriction of " chemotherapeutics ": (i) effect is anti-for the hormonal action in adjusting or inhibition tumour Hormone agents such as antiestrogenic and selective estrogen receptor modulators (SERM), for example including tamosifen (includingCitric acid tamosifen), Raloxifene, Droloxifene, 4- hydroxy tamoxifen, Trioxifene, that Lip river Former times sweet smell (keoxifene), LY117018, Onapristone and(citric acid toremifene)；(ii) inhibit enzyme The aromatase inhibitor of aromatase enzyme, adjusting result from adrenal estrogen such as such as 4 (5)-imidazoles, aminoglutethimide,(megestrol acetate),(Exemestane；Pfizer), Formestane (formestanie), Fadrozole,(Vorozole),(Letrozole；Novartis) and(Anastrozole；AstraZeneca)；(iii) antiandrogen such as Drogenil, Nilutamide, than card Shandong Amine, Leuprorelin and Goserelin；And troxacitabine (1,3- dioxolane nucleoside analogue of cytosine)；(iv) protein swashs Enzyme inhibitor such as mek inhibitor (WO 2007/044515)；(v) lipid kinase inhibitors；(vi) antisense oligonucleotides, especially Inhibit those of gene such as PKC- α, Raf and the H-Ras expression of signal path impacted in abnormal cell proliferation, benefit such as difficult to understand Write from memory gloomy (oblimersen) (Genta Inc.)；(vii) ribozyme such as vegf expression inhibitor (example Such as,) and HER2 expression inhibiting agent；(viii) vaccine such as gene therapy vaccine, such asWithrIL-2；Topology is different 1 inhibitor of structure enzyme is such asrmRH；(ix) anti-angiogenic producing agent such as shellfish cuts down pearl Monoclonal antibody () and the pharmaceutically acceptable salt of any of the above, acid and derivative Genentech.
Further include " chemotherapeutics " restriction in be therapeutic antibodies such as alemtuzumab (Campath), Avastin (Genentech)；Cetuximab (Imclone)；Victibix (Amgen), Rituximab (Genentech/Biogen Idec), method difficult to understand wood Monoclonal antibody (GSK), handkerchief trastuzumab (PERJETATM, OMNITARGTW, 2C4, Genentech), toltrazuril Monoclonal antibody (Genentech), tositumomab (Bexxar, Corixia) and antibody drug conjugate, Ji Trastuzumab (gemtuzumab ozogamicin) (Wyeth)。
With the Humanized monoclonal antibodies that the treatment potentiality as chemotherapeutics is combined with conjugate of the invention include: Ah Logical sequence monoclonal antibody, A Bozhu monoclonal antibody, A Saizhu monoclonal antibody, atlizumab, Ba Pinzhu monoclonal antibody (bapineuzumab), Avastin, not Than cutting down pearl monoclonal antibody (bivatuzumab mertansine), not bank trastuzumab (cantuzumab mertansine), Seeley pearl Monoclonal antibody, match trastuzumab (certolizumab pegol), cidfusituzumab, cidtuzumab, daclizumab, Yi Ku The sharp pearl monoclonal antibody of beautiful monoclonal antibody, efalizumab, epratuzumab, strategic point, felvizumab, fragrant trastuzumab (Fontolizumab), lucky trastuzumab, English trastuzumab (Inotuzumab Ozogamicin), her monoclonal antibody, draw Bei Zhudan Anti-, lintuzumab, matuzumab, mepolizumab, Mo Weizhu monoclonal antibody, motovizumab, natalizumab, the appropriate pearl of Buddhist nun Monoclonal antibody, nolovizumab, numavizumab, auspicious pearl monoclonal antibody (ocrelizumab) difficult to understand, omalizumab, palivizumab, pa Examine pearl monoclonal antibody, pecfusituzumab, pectuzumab, handkerchief trastuzumab, training gram pearl monoclonal antibody, ralivizumab, Lei Zhudan Anti-, reslivizumab, Rayleigh pearl monoclonal antibody, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, Seeley pearl Monoclonal antibody (Siplizumab), Suo Tuzhu monoclonal antibody, his pearl monoclonal antibody (tacatuzumab tetraxetan), he spend pearl monoclonal antibody, his benefit Pearl monoclonal antibody, special non-pearl monoclonal antibody, tower Xidan is anti-, holds in the palm sharp pearl monoclonal antibody (toralizumab), trastuzumab, tucotuzumab Celmoleukin, tucusituzumab, umavizumab, black pearl monoclonal antibody and Wei Xi pearl monoclonal antibody (visilizumab).
According to the invention and pharmaceutical composition used according to the present invention is removed active constituent (that is, in conjunction with compounds) It in addition, can also include pharmaceutical excipient, carrier, buffer, stabilizer or other substances well known to those skilled in the art.This The substance of sample should be effect that is nontoxic, and should not interfere with active constituent.The definite property of carrier or other substances will depend on In giving approach, this is given approach and can be orally or through injection (such as skin, subcutaneous or intravenous).
It can be tablet, capsule, powder or liquid form for the oral pharmaceutical composition given.Tablet may include Solid carrier or adjuvant.Composition of liquid medicine generally comprises liquid-carrier such as water, petroleum (petroleum), animal or plant Oil, mineral oil or synthetic oil.It may include normal saline solution, dextrose or other sugar juices or glycols such as ethylene glycol, third Glycol or polyethylene glycol.Capsule may include solid carrier such as gelatin.
For intravenous, skin or subcutaneous injection, or the injection at painful position, active component will have parenteral The form of acceptable aqueous solution is pyrogen-free and has suitable pH, isotonicity and stability.Those skilled in the art It is able to use for example, isotonic vehicle such as sodium chloride injection, ringer's injection, lactated ringer's injection are prepared well Suitable solution.It as needed, may include preservative, stabilizer, buffer, antioxidant and/or other additives.
Preparation
Although can be used alone (for example, giving) in conjunction with compounds, often preferably make it as composition Or preparation exists.
In one embodiment, composition is comprising combination compounds described herein and pharmaceutically acceptable The pharmaceutical composition (for example, preparation, preparation, drug) of carrier, diluent or excipient.
In one embodiment, composition be comprising at least one combination compounds described herein and it is a kind of or The pharmaceutical composition of a variety of pharmaceutically acceptable ingredients well known to those skilled in the art, which includes but is not limited to pharmacy Upper acceptable carrier, diluent, excipient, adjuvant, filler, buffer, preservative, antioxidant, lubricant, stabilizer, Solubilizer, surfactant (for example, wetting agent), screening agent, colorant, flavoring agent and sweetener.
In one embodiment, composition further includes other activating agents, for example, other therapeutic agents or prophylactic.
Suitable carrier, diluent, excipient etc. can be found in standard pharmaceutical text.See, e.g.,Handbook of Pharmaceutical Additives,2nd Edition(eds.M.Ash and I.Ash),2001(Synapse Information Resources,Inc.,Endicott,New York,USA),Remington's Pharmaceutical Sciences,20th edition,pub.Lippincott,Williams&Wilkins,2000；WithHandbook of Pharmaceutical Excipients,2nd edition,1994。
Another aspect of the present invention relates to the methods of preparation pharmaceutical composition, including at least one that will be limited herein [11C]-labelled with radioisotope conjugate or combine class compound and it is one or more it is well known to those skilled in the art its He mixes pharmaceutically acceptable ingredient, for example, carrier, diluent, excipient etc..If being formulated as discrete unit (for example, tablet etc.), then each unit includes the reactive compound of predetermined amount (dosage).
The term as used herein " pharmaceutically acceptable " is related to compound, ingredient, material, composition, dosage form etc., Reasonable medical judgment scope is interior, excessive suitable for contacting without with the tissue of the subject (for example, mankind) discussed Toxicity, stimulation, allergic reaction or other problems or complication, and match with reasonable interests/Hazard ratio.Every kind of carrier, dilution Agent, excipient etc. also must be " acceptable " in the sense that compatible with the other compositions of preparation.
Preparation can be prepared by any method known to pharmaceutical field.Such method includes making reactive compound and structure At the carrier-bound step of one or more auxiliary elements.In general, preparation is by making reactive compound and carrier (example Such as, liquid-carrier, fine solid carrier etc.) it is uniform closely combine, make product shaping if necessary then to prepare.
Preparation can be prepared as discharging quickly or at a slow speed；Immediately, delay, instant or slow release；Or their combination.
Preparation suitable for parenterally giving (for example, passing through injection) includes aqueous or non-aqueous, isotonic, apyrogeneity , sterile liquid (such as solution, suspension), wherein active constituent be dissolved, suspend or otherwise provide (for example, In liposome or other particles).In addition this liquid can contain other pharmaceutically acceptable ingredient such as antioxidants, buffering Agent, preservative, stabilizer, bacteriostatic agent, suspending agent, thickener and blood (or other dependent bodies for making preparation and intended recipient Liquid) isotonic solute.The example of excipient includes, for example, water, alcohol, polyalcohol, glycerol, vegetable oil etc..For such preparation The example of suitable isotonic vehicle includes sodium chloride injection, ringer's solution (Ringer's Solution) or lactated Ringer note Penetrate liquid.In general, the concentration of active constituent is about 1ng/ml to about 10 μ g/mL, for example, about 10ng/mL to about 1 μ g/mL in liquid. Said preparation may be present in unit dose or multi-dose sealing container, such as in ampoule and phial, and (can freeze in freeze-drying It is dry) under the conditions of store, it is only necessary to sterile liquid carrier such as water for injection is being added before use.Can by aseptic powdery, particle and Tablet prepares injection solution and suspension on the spot.
Dosage
It will be apparent to a skilled person that in conjunction with compounds and including the composition for combining compounds Suitable dosage can be different because of patient.The level for making treatment benefit and any risk or harmful secondary will be involved by determining optimal dose generally Effect balance.The dosage level of selection will depend on Multiple factors, including but not limited to the activity of specific compound, give way Diameter, excretion time, the duration for the treatment of, other drugs, compound and/or the object being applied in combination for giving time, compound Matter, the seriousness of the patient's condition and species, gender, age, weight, the patient's condition, general health and the medical history of patient.The amount of compound It finally will cautiously be determined by doctor, animal doctor or clinician with approach of giving, but in general, by selection dosage to act on Position, which reaches, realizes the desired local concentration acted on without will cause material injury or harmful side effect.
During the entire course for the treatment of, can with single dose, it is continuously or intermittently (for example, with appropriate time interval divided dose) real Now give.Determine that the most effective means and the method for dosage given are well known to those skilled in the art, and will be with being used to treat The preparation of method, therapy purpose, (a variety of) target cell treated and the difference of the subject treated and change.Treatment can be used Doctor, the dosage level of animal doctor or clinician's selection and mode carry out single or multiple give.
In general, the suitable dose of reactive compound is that (more generally about 1 μ g is to about in about 100 μ g to about 25mg In the range of 10mg)/kg of body's body weight/day.In the case where the compound is salt, ester, amide, prodrug etc., based on mother Body compound calculates administered dose, so the proportional increase of actual weight used.
In one embodiment, reactive compound is given to human patients according to following dosage: about 100mg, 3 times a day.
In one embodiment, reactive compound is given to human patients according to following dosage: about 150mg, 2 times a day.
In one embodiment, reactive compound is given to human patients according to following dosage: about 200mg, 2 times a day.
However, in one embodiment, will be given in conjunction with compounds to human patients according to following dosage: about 50 or about 75mg, daily 3 or 4 times.
In one embodiment, will be given to human patients in conjunction with compounds according to following dosage: about 100 or About 125mg, 2 times a day.
Dosage described above can be adapted for conjugate (comprising PBD part and to antibody connector) or be suitable for provide PBD compound effective quantity, for example, the amount of the compound discharged after joint cutting.
For preventing or treating disease, the suitable dosage of ADC of the invention will depend on disease defined above to be treated Sick type, the severity of disease and situation (no matter giving molecule for prevention or therapeutic purposes), prior treatment, patient Clinical medical history and to the reaction of antibody and the judgement of attending physician.Once or by a series of treatments by molecule suitably give It gives to patient.Depending on the type and severity of disease, no matter, such as individually given or by one or many by continuous Infusion, the molecule of about 1 μ g/kg to 15mg/kg (for example, 0.1-20mg/kg) is for giving to the initial candidate dosage of patient. Common daily dosage can be in about 1 μ g/kg to 100mg/kg or more, this depends on above-mentioned factor.It gives To patient ADC exemplary dose in the range of about 0.1 to about 10mg/kg patient weight.For several days or longer heavy It gives again, depends on illness, treatment, which continues to, realizes that the expectation of disease symptoms inhibits.Exemplary dose scheme includes giving about The case where 4mg/kg initial drugloading rate, then weekly, every two weeks or the ADC of the every three weeks other dosage of addition.Other dosages It can be useful.The progress of the therapy is easy to monitor by routine techniques and measuring method.
Treatment
In the case where treating illness term as used herein " treatment " be usually directed to no matter mankind or animal (for example, Veterinary application) treatment and therapy, wherein wherein realize some desired curative effects, for example, the inhibition to ongoing disease, and And including development speed reduce, development speed halves, illness is restored, illness improves and illness rehabilitation.It further include as preventative The treatment of measure (that is, prevent, prevent).
Term as used herein " therapeutically effective amount " is related to reactive compound or the substance comprising reactive compound, combination The amount of object or dosage form is effective and closes when being given according to desired therapeutic scheme for generating some desired curative effects Interests/Hazard ratio of reason is suitable.
Similarly, term as used herein " prevention effective dose " is related to reactive compound or the object comprising reactive compound The amount of matter, composition or dosage form, when being given according to desired therapeutic scheme, desired preventive effects some for generation are Effectively, suitable with reasonable interests/Hazard ratio.
The preparation of drug conjugates
Can by number of ways well known by persons skilled in the art, using organic chemical reactions, condition and reagent, including The nucleophilic group of antibody is reacted with agent-linker reagent, prepares antibody drug conjugate.This method can be used to prepare this The antibody-drug conjugates of invention.
Nucleophilic group on antibody includes but is not limited to side chain thiol moiety, for example, cysteine.Thiol group is nucleophilic And can react to form covalent bond with the electrophilic group (such as those of present invention) on junction portion.Certain antibody have can Disulphide in the chain of reduction, that is, cysteine bridge.By with reducing agent such as DTT (Ke Lailanshi reagent (Cleland's), two Thio threitol) or TCEP (three (2- carboxyethyl) hydrogen phosphide hydrochlorides；Getz et al(1999)Anal.Biochem.Vol 273:73-80；Soltec Ventures, Beverly, MA) processing can make the combination of antibody and linker reagents become easily anti- It answers.Therefore, the last cysteine disulphide bridges of every theory will form two reactive nucleophilic thiol bodies.By generating amine to mercaptan The lysine of conversion is reacted with 2- imino group thiophene (Te Laoteshi (Traut ' s) reagent), other nucleophilic groups can be drawn Enter into antibody.
Subject/patient
Subject/patient can be animal, mammal, placental mammals, marsupial (for example, kangaroo, wombat), Monotreme (for example, platypus), rodent (for example, cavy, hamster, rat, mouse), murine are (for example, small Mouse), lagomorph (for example, rabbit), birds (for example, bird), canid (for example, domesticated dog), felid is (for example, family Cat), equid (for example, horse), porcine animals (for example, domestic pig), sheep class (for example, sheep), bovid is (for example, milk Ox), primate, apes (for example, monkey or anthropoid), monkey (for example, marmoset, baboon), anthropoid (for example, gorilla, Chimpanzee, orangutan, gibbon) or the mankind.
In addition, subject/patient can be any form of its development, such as fetus.In a preferred embodiment, by Examination person/patient is the mankind.
Further preference
It preferably can be adapted for all aspects of invention described above below or can be related to single aspect.This preferably can be with It is combined with any combination.
In some embodiments, R6’、R7’、R9’And Y ' preferably respectively with R6、R7、R9It is identical with Y.
Dimer connection
Y and Y ' are preferably O.
R " is suitably without the C of substituent group3-7Alkylidene.It is highly preferred that R " is C3、C5Or C7Alkylidene.Most preferably, R " It is C3Or C5Alkylidene.
R6To R9
R9Preferably H.
R6It is preferably selected from H, OH, OR, SH, NH2, nitro and halogen, and more preferably H or halogen, and most preferably H.
R7It is preferably selected from H, OH, OR, SH, SR, NH2, NHR, NRR ' and halogen, and be more preferably independently selected from H, OH and OR, wherein R is preferably selected from optionally substituted C1-7Alkyl, C3-10Heterocycle and C5-10Aryl.R can more preferably can with or not It can be with substituted C1-4Alkyl.Interested substituent group is C5-6Aryl (such as phenyl).Particularly preferred at 7 positions takes Dai Ji is OMe and OCH2Ph.Other particularly interesting substituent groups are dimethylamino (i.e.-NMe2)；-(OC2H4)qOMe, Wherein q is 0 to 2；Nitrogenous C6Heterocycle, including morpholino, piperidyl and N- methyl-piperazinyl group.
These preferences are respectively suitable for R9’、R6’And R7’。
R12
As C2 ' and C3 ' between there are when double bond, R12It is selected from:
(a)C5-10Aryl, optionally by selected from including below group one or more substituent groups replace: halogen, nitro, Cyano, ether, C1-7Alkyl, C3-7Heterocycle and double-oxygroup-C1-3Alkylidene；
(b)C1-5Radical of saturated aliphatic alkyl；
(c)C3-6Saturated cyclic alkyls；
(d)Wherein R21、R22And R23It is each independently selected from H, C1-3Saturated alkyl, C2-3Alkenyl, C2-3Alkynes Base and cyclopropyl, wherein in R12The sum of carbon atom is not more than 5 in group；
(e)Wherein R25aAnd R25bIn one be H and another is selected from: (it is by selected from halogenated to phenyl Methyl, the group of methoxyl group are optionally substituted)；Pyridyl group；And thiophenyl；And
(f)Wherein R24It is selected from: H；C1-3Saturated alkyl；C2-3Alkenyl；C2-3Alkynyl；Cyclopropyl；Phenyl (its It is by optionally substituted selected from halogenated methyl, the group of methoxyl group)；Pyridyl group；And thiophenyl.
Work as R12It is C5-10When aryl, it can be C5-7Aryl.C5-7Aryl can be phenyl or C5-7Heteroaryl, such as furan It mutters base, thiophenyl and pyridyl group.In some embodiments, R12Preferably phenyl.In other embodiments, R12Preferably Thiophenyl, for example, thiophene -2- base and thiene-3-yl.
Work as R12It is C5-10When aryl, it can be C8-10Aryl, such as quinolyl or isoquinolyl.Quinolyl or isoquinolin Base can be incorporated into PBD core by any available ring position.For example, quinolyl can be quinoline -2- base, quinoline -3- base, Quinolyl-4, quinoline -5- base, quinoline -6- base, quinoline -7- base and quinoline-8-yl.Wherein, quinoline -3- base and quinoline -6- base It can be preferred.Isoquinolyl can be isoquinolyl-1, isoquinolin -3- base, isoquinolin -4- base, isoquinolin -5- base, different Quinoline -6- base, isoquinolin -7- base and isoquinolin -8- base.Wherein, isoquinolin -3- base and isoquinolin -6- base can be preferably.
Work as R12It is C5-10When aryl, it can have any amount of substituent group.It preferably has 1 to 3 substituent group, In 1 and 2 substituent group be more preferred, and mono-substituted group is most preferred.Substituent group can be in any position.
In R12It is C5-7In the case where aryl, monosubstituted base preferably on annular atom, not adjacent to compound The key of remainder, that is, it is preferably β or γ with the key of the remainder of compound.Therefore, in C5-7Aryl is phenyl In the case where, substituent group is more preferably aligning preferably in meta or para position.
In R12It is C8-10In the case where aryl, such as quinolyl or isoquinolyl, it can be in quinoline or isoquinolin ring Any amount of substituent group is had at any position.In some embodiments, it has one, two or three substituent group, And these substituent groups can be upper (if more than one substituent group) in nearside or distal loop or both.
R12Substituent group works as R12It is C5-10When aryl group
Work as R12It is C5-10When aryl, if in R12On substituent group be halogen, then it is preferably F or Cl, more preferable Cl.
Work as R12It is C5-10When aryl, if in R12On substituent group be ether, then it can be alkane in some embodiments Oxygroup, for example, C1-7Alkoxy (such as methoxyl group, ethyoxyl), or it can be C in some embodiments5-7Aryloxy group (example Such as phenoxy group, pyridine oxygroup, furans oxygroup).Alkoxy itself can be further substituted, such as by amino (such as dimethyl Amino).
Work as R12It is C5-10When aryl, if in R12On substituent group be C1-7Alkyl, it can be preferably C1-4Alkyl (such as Methyl, ethyl, propyl, butyl).
Work as R12It is C5-10When aryl, if in R12On substituent group be C3-7Heterocycle, then it can in some embodiments To be C6Nitrogen heterocycle, such as morpholino, thiomorpholine generation, piperidyl, piperazinyl.These groups can be via nitrogen-atoms knot Together in the remainder of the part PBD.These groups can be further substituted, for example, by C1-4Alkyl.If C6Nitrogen-containing heterocycle Base is piperazinyl, then the further substituent group can be on the second nitrogen ring atom.
Work as R12It is C5-10When aryl, if in R12On substituent group be double-oxygroup-C1-3Alkylidene, this is preferably double-oxygen Base-methylene or double-oxygroup-ethylidene.
Work as R12It is C5-10When aryl, if R12On substituent group be ester, then it is preferably methyl ester or ethyl ester.
Work as R12It is C5-10When aryl, particularly preferred substituent group include methoxyl group, ethyoxyl, fluorine-based, chloro, cyano, it is double- Oxygroup-methylene, methyl-piperazinyl group, morpholino and methyl-phenylsulfanyl.For R12Other particularly preferred substituent groups be two Methylamino propoxyl group and carboxyl.
Work as R12It is C5-10When aryl, particularly preferred substituted R12Group includes but is not limited to: 4- methoxyl group-phenyl, 3- Methoxyphenyl, 4- ethyoxyl-phenyl, 3- ethyoxyl-phenyl, 4- fluoro-phenyl, the chloro- phenyl of 4-, 3,4- dioxymethylene-benzene Base, 4- methylphenyl-sulfanyl, 4- cyano-phenyl, 4- Phenoxyphenyl, quinoline -3- base and quinoline -6- base, isoquinolin -3- base and different Quinoline -6- base, 2- thienyl, 2- furyl, methoxyl group naphthalene and naphthalene.Alternatively possible substituted R12Group is 4- nitro Phenyl.R of special interest12Group includes 4- (4- methylpiperazine-1-yl) phenyl and 3,4- dioxygen methylene-phenyl.
Work as R12It is C1-5When radical of saturated aliphatic alkyl, it can be methyl, ethyl, propyl, butyl or amyl.In some implementations In mode, it can be methyl, ethyl or propyl (n-pentyl or isopropyl).These embodiments it is some in, it can be with It is methyl.In other embodiments, it can be butyl or amyl, can be linear chain or branched chain.
Work as R12It is C3-6When saturated cyclic alkyls, it can be cyclopropyl, cyclobutyl, cyclopenta or cyclohexyl.In some implementations In mode, it can be cyclopropyl.
Work as R12It isWhen, R21、R22And R23It is each independently selected from H, C1-3Saturated alkyl, C2-3Alkenyl, C2-3 Alkynyl and cyclopropyl, wherein in R12The sum of carbon atom in group is not more than 5.In some embodiments, in R12In group Carbon atom sum no more than 4 or be not more than 3.
In some embodiments, R21、R22And R23One of be H, and other two kinds of groups be selected from H, C1-3It is saturated alkane Base, C2-3Alkenyl, C2-3Alkynyl and cyclopropyl.
In other embodiments, R21、R22And R23In two kinds be H, and a kind of other groups are selected from H, C1-3It is saturated alkane Base, C2-3Alkenyl, C2-3Alkynyl and cyclopropyl.
It in some embodiments, is not the group of H selected from methyl and ethyl.These embodiments it is some in, no The group for being H is methyl.
In some embodiments, R21It is hydrogen.
In some embodiments, R22It is hydrogen.
In some embodiments, R23It is hydrogen.
In some embodiments, R21And R22It is H.
In some embodiments, R21And R23It is H.
In some embodiments, R22And R23It is H.
Particularly interesting R12Group is:
Work as R12It isWhen, R25aAnd R25bIn one be hydrogen, and another is selected from: (it is by being selected to phenyl Halogen, methyl, the group of methoxyl group are optionally substituted)；Pyridyl group；And thiophenyl.It in some embodiments, is not the base of H Group is optionally substituted phenyl.If the optional substituent group of phenyl is halogen, it is preferably fluorine.In some embodiments, Phenyl is unsubstituted.
Work as R12It isWhen, R24It is selected from: H；C1-3Saturated alkyl；C2-3Alkenyl；C2-3Alkynyl；Cyclopropyl；Phenyl (it is by optionally substituted selected from halogenated methyl, the group of methoxyl group)；Pyridyl group；And thiophenyl.If phenyl is optional Substituent group is halogen, then it is preferably fluorine.In some embodiments, phenyl is unsubstituted.
In some embodiments, R24Selected from H, methyl, ethyl, vinyl and acetenyl.The one of these embodiments In a little, R24Selected from H and methyl.
As C2 ' and C3 ' between there are when singly-bound,
R12It isWherein, R26aAnd R26bIndependently selected from H, F, C1-4Saturated alkyl, C2-3Alkenyl, the alkyl and Alkenyl group is optionally selected from C1-4Alkyl amino and C1-4The group of Arrcostab replaces；Alternatively, working as R26aAnd R26bIn one When being H, another is selected from nitrile and C1-4Arrcostab；
In some embodiments, it may be preferable that R26aAnd R26bIt is all H.
In other embodiments, preferably R26aAnd R26bIt is all methyl.
In further embodiment, preferably R26aAnd R26bIn one be H and another is selected from C1-4It is saturated alkane Base, C2-3Alkenyl, wherein alkyl and alkenyl group are optionally substituted.It, can be further in these further embodiments It is preferred that whether the group of H is selected from methyl and ethyl.
R2
Above for R12Preference be equally applicable to R2。
R22
In some embodiments, R22It is Formula II a.
When it is Formula II a, R22In A can be phenyl group or C5-7Heteroaryl groups, such as furyl, thiophenyl And pyridyl group.In some embodiments, A is preferably phenyl.
Q2- X can be in C5-7On any available annular atom of aryl group, but preferably in the remainder with compound On the non-conterminous annular atom of key divided, that is, preferably at β or γ of the key of the remainder of compound.Therefore, work as C5-7 When aryl group (A) is phenyl, substituent group (Q2- X) preferably in meta or para position, and more preferably aligning.
In some embodiments, Q1It is singly-bound.In these embodiments, Q2Selected from singly-bound and-Z- (CH2)n, In, Z is selected from singly-bound, O, S and NH, and n is 1 to 3.These embodiments it is some in, Q2It is singly-bound.In other embodiment party In formula, Q2It is-Z- (CH2)n-.In these embodiments, Z can be O or S, and n can be 1 or n can be 2.At these In the others of embodiment, Z can be singly-bound, and n can be 1.
In other embodiments, Q1It is-CH=CH-.
In other embodiments, R22It is Formula II b.In these embodiments, RC1、RC2And RC3Independently selected from H and not Substituted C1-2Alkyl.In some preferred embodiments, RC1、RC2And RC3It is all H.In other embodiments, RC1、RC2 And RC3It is all methyl.In some embodiments, RC1、RC2And RC3Independently selected from H and methyl.
X is selected from the group comprising list below: O-RL2’、S-RL2’、CO2-RL2’、CO-RL2’, NH-C (=O)-RL2’、 NHNH-RL2’、CONHNH-RL2’、 NRNRL2’, wherein RNSelected from including H and C1-4 The group of alkyl.X can be preferred that: OH, SH, CO2H ,-N=C=O or NHRN, and can be more preferably: O-RL2’、S- RL2’、CO2-RL2’,-NH-C (=O)-RL2’Or NH-RL2’.Particularly preferred group includes: O-RL2’、S-RL2’And NH-RL2’, NH- RL2’It is most preferred group.
In some embodiments, R22It is Formula II c.In these embodiments, preferably Q is NRN-RL2’.At other In embodiment, Q is O-RL2’.In further embodiment, Q is S-RL2’。RNIt is preferably chosen from H and methyl.Some In embodiment, RNIt is H.In other embodiments, RNIt is methyl.
In some embodiments, R22It can be-A-CH2- X and-A-X.In these embodiments, X can be O- RL2’、S-RL2’、CO2-RL2’、CO-RL2’And NH-RL2’.In particularly preferred embodiments, X can be NH-RL2’。
R10、R11
In some embodiments, R10And R11The double bond being formed together between the nitrogen and carbon atom that they are connected.
In some embodiments, R11It is OH.
In some embodiments, R11It is OMe.
In some embodiments, R11It is SOzM, wherein z be 2 or 3 and M be unit price it is pharmaceutically acceptable sun from Son.
R11a
In some embodiments, R11aIt is OH.
In some embodiments, R11aIt is OMe.
In some embodiments, R11aIt is SOzM, wherein z is the pharmaceutically acceptable sun that 2 or 3 and M is unit price Ion.
R20、R21
In some embodiments, R20And R21The double bond being formed together between the nitrogen and carbon atom that they are connected.
In some embodiments, R20It is H.
In some embodiments, R20It is RC。
In some embodiments, R21It is OH.
In some embodiments, R21It is OMe.
In some embodiments, R21It is SOzM, wherein z be 2 or 3 and M be unit price it is pharmaceutically acceptable sun from Son.
R30、R31
In some embodiments, R30And R31The double bond being formed together between the nitrogen and carbon atom that they are connected.
In some embodiments, R31It is OH.
In some embodiments, R31It is OMe.
In some embodiments, R31It is SOzM, wherein z be 2 or 3 and M be unit price it is pharmaceutically acceptable sun from Son.
M and z
Preferably, M is the cation of the pharmaceutical acceptable of unit price, and more preferably Na+。
Z is preferably 3.
The D that can have Formulas I a preferably in combination with object of the first aspect of the present inventionL:
Wherein,
RL1’、R20And R21It is as defined above；
N is 1 or 3；
R1aIt is methyl or phenyl；And
R2aIt is selected from:
(a)
(b)
(c)
(d)
(e)
(f)
(g)With
(h)
The D that can have Formulas I b preferably in combination with object of the first aspect of the present inventionL:
Wherein,
RL1’、R20And R21As defined above；
N is 1 or 3；And
R1aIt is methyl or phenyl.
The D that can have Formulas I c preferably in combination with object of the first aspect of the present inventionL:
Wherein, RL2’、R10、R11、R30And R31As defined above,
N is 1 or 3；
R12aIt is selected from:
(a)
(b)
(c)
(d)
(e)
(f)
(g)With
(h)
Meta or para position of the amino group in phenyl group.
The D that can have Formulas I d preferably in combination with object of the first aspect of the present inventionL:
Wherein, RL2’、R10、R11、R30And R31As defined above,
N is 1 or 3；
R1aIt is methyl or phenyl；
R12aIt is selected from:
(a)
(b)
(c)
(d)
(e)
(f)
(g)With
(h)
The D that can have Formulas I e preferably in combination with object of the first aspect of the present inventionL:
Wherein, RL2’、R10、R11、R30And R31As defined above,
N is 1 or 3；
R1aIt is methyl or phenyl；
R12aIt is selected from:
(a)
(b)
(c)
(d)
(e)
(f)
(g)With
(h)
Embodiment
General Experimental Procedures
As unit of measuring optical activity on 220 polarimeter of ADP (Bellingham Stanley Ltd.) and by g/100mL To provide concentration (c).Fusing point is measured using numeral melting point instrument (Electrothermal).Use Perkin-Elmer Spectrum 1000FT IR spectrometer is composed to record IR.At 300k, using Bruker Avance NMR spectrometer, respectively It is obtained at 400 and 100MHz1H and13C H NMR spectroscopy.Chemical shift is reported relative to TMS (δ=0.0ppm), and by signal designation For s (unimodal), d (bimodal), t (triplet), dt (double triplets), dd (bimodal is bimodal), ddd (bimodal double doublet) or m (multiplet), wherein providing coupling constant so that hertz (Hz) is unit.Using being connected to Waters 2996PDA's Waters Micromass ZQ instrument on Waters 2695HPLC collects mass spectrum (MS) data.The Waters used Micromass ZQ parameter is: capillary (kV), 3.38；It bores voltage (V), 35；Extractor (V), 3.0；Source temperature (DEG C), 100； Desolvation temperature (DEG C), 200；Coning tower tray speed (L/h), 50；Desolvation flow velocity (L/h), 250.The borosilicate coated using metal Glass tip introduces the sample into instrument, with positive W mode records high-resolution on Waters Micromass QTOF Global Rate mass spectrum (HRMS) data.In silica gel aluminium sheet (Merck 60, F254) on carry out thin-layer chromatography (TLC), and utilize silica gel (Merck 60,230-400 mesh ASTM) Lai Jinhang flash chromatography.Except HOBt (NovaBiochem) and supported reagents (Argonaut) it Outside, every other chemicals and solvent are purchased from Sigma-Aldrich and as that supplies is used without further purifying.Having Under the conditions of appropriate desiccant is existing, under a dry nitrogen atmosphere, anhydrous solvent is prepared by distilling, and be stored inPoint On son sieve or sodium wire.Petroleum ether refers to the fraction to boil at 40-60 DEG C.
Common LC/MS condition:
Method 1 (otherwise default method uses unless otherwise noted)
HPLC (Waters is run using the mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%) Alliance 2695).Gradient: initial composition 5%B continues 1.0 minutes, then, in 3 minutes, is increased to 95% from 5%B B.Above-mentioned composition is maintained at 95%B lower 0.1 minute, 5%B is then returned in 0.03 minute and keeps 0.87min.Total gradient Runing time is equal to 5 minutes.
Method 2
HPLC (Waters is carried out using the mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%) Alliance 2695).Gradient: initial composition 5%B continues 1.0 minutes, then, in 2.5 minutes, is increased to from 5%B 95%B.Above-mentioned composition is maintained at 95%B lower 0.5 minute, 5%B is then returned in 0.1 minute and keeps 0.9min.Total ladder Runing time is spent to be equal to 5 minutes.
For each method
Flow velocity 3.0mL/min separates 400 μ L via zero dead volume T shape pipe (it is passed through mass spectrograph).Wavelength detecting model It encloses: 220 to 400nm.Function type: diode array (535 scanning).Column: Onyx Monolithic C1850×4.60mm。
It is as follows that reverse phase rapidly purifies condition: carrying out flash purification system using the mobile phase of water (A) and acetonitrile (B) (Varian 971-Fp).Gradient: under 20C.V (column volume), initial composition 5%B, then from 5%B to 70% in 60C.V B.The composition of 95%B is kept into 15C.V., 5%B is then returned in 5C.V., and be maintained at 5%B and continue 10C.V..Total gradient Runing time is equal to 120C.V.Flow velocity: 6.0mL/min.Wavelength detection range: 254nm.Column: Agilent AX1372-1SF10- 5.5gC8。
Preparative HPLC: reverse phase ultra high efficiency is carried out on the 5 μ C-18 column of Phenomenex Gemini NX of following size Liquid chromatography (UPLC).150 × 4.6mm is used to analyze and 150 × 21.20mm is used to prepare work.Pass through gradient condition Execute all UPLC experiments.Eluent is the solvent A (H with 0.1% fumaric acid2O) and solvent B (has 0.1% fumaric acid CH3CN).The flow velocity used is that 1.0ml/min is used to analyze and 20.0ml/min is used to prepare type HPLC.In 254nm and It is detected at 280nm.
The synthesis of intermediate 12
(a) 1 ', 3 '-bis- [2- methoxyl group -4- (methoxycarbonyl) phenoxy group] propane (3)
Under 0-5 DEG C (ice/acetone), in a nitrogen atmosphere, by azo-2-carboxylic acid's diisopropyl ester within the 60min period (71.3mL, 73.2g, 362mmol) is added dropwise to vanillic acid methyl esters 2 (60.0g, 329mmol) and Ph3P (129.4g, 494mmol) in the solution of the upper belt stirring in anhydrous THF (800mL).Permission is stirred to react mixture other 1 at 0-5 DEG C Hour, after which time, 1,3-PD (11.4mL, 12.0g, 158mmol) is added dropwise within the 20min period in THF Solution in (12mL).Allow for reaction mixture to be warming up to room temperature, and stirs 5 days.It is collected by vacuum filtration White depositions 3 are washed with THF and are dried in vacuum desiccator to constant weight.Yield=54.7g (it is based on 1,3-PD, 84%).Purity satisfaction, LC/MS (3.20min (ES+) m/z (relative intensity) 427 ([M+Na]+., 10)；1H NMR (400MHz, CDCl3) δ 7.64 (dd, 2H, J=1.8,8.3Hz), 7.54 (d, 2H, J=1.8Hz), 6.93 (d, 2H, J= 8.5Hz), 4.30 (t, 4H, J=6.1Hz), 3.90 (s, 6H), 3.89 (s, 6H), 2.40 (p, 2H, J=6.0Hz).
(b) 1 ', 3 '-bis- [2- methoxyl group -4- (methoxycarbonyl) -5- nitro-phenoxy] propane (4)
Under 0-5 DEG C (ice/acetone), by solid Cu (NO3)2·3H2O (81.5g, 337.5mmol) be added slowly to it is double- Ester 3 (54.7g, 135mmol) is in the slurry of the top band stirring in acetic anhydride (650mL).Permission is stirred to react mixed at 0-5 DEG C It closes object 1 hour, then it is allowed to be warming up to room temperature.At this stage, slight exotherm (about 40-50 DEG C) is observed, along with mixing Object retrogradation and NO2Release.Other acetic anhydride (300mL) is added, and it is small to allow to be stirred at room temperature reaction mixture 16 When.Reaction mixture is poured on ice (~1.5L), and stirs and it is allowed to be back to room temperature.It is collected by vacuum filtration Obtained yellow mercury oxide, and dry in drier to provide desired double -4 yellow solid of nitro compound.Yield= 66.7g (100%).Purity satisfaction, LC/MS (3.25min (ES+) m/z (relative intensity) 517 ([M+Na]+., 40)；1H NMR (400MHz, CDCl3) δ 7.49 (s, 2H), 7.06 (s, 2H), 4.32 (t, 4H, J=6.0Hz), 3.95 (s, 6H), 3.90 (s, 6H), 2.45-2.40 (m, 2H).
(c) 1 ', 3 '-bis- (4- carboxyl -2- methoxyl group -5- nitro-phenoxy) propane (5)
The slurry of methyl esters 4 (66.7g, 135mmol) in THF (700mL) is handled with 1N NaOH (700mL), and is allowed It is vigorously mixed at room temperature for reaction mixture.After stirring 4 days, which becomes dark solution, undergoes the solution under reduced pressure Rotary evaporation is to remove THF.With dense HCl be acidified it is aqueous remain to pH 1, and collect colorless precipitation 5, Yi Ji It is thoroughly dried in vacuum drying oven (50 DEG C).Yield=54.5g (87%).Purity satisfaction, LC/MS (2.65min (ES+) m/z (relative intensity) 489 ([M+Na]+.,30)；1H NMR (400MHz, DMSO-d6) δ 7.62 (s, 2H), 7.30 (s, 2H), 4.29 (t, 4H, J=6.0Hz), 3.85 (s, 6H), 2.30-2.26 (m, 2H).
(d) 1,1 '-[[(propane -1,3- diyl) dioxy] is bis- [(5- methoxyl group -2- nitro -1,4- phenylene) carbonyl]] are double [(2S, 4R)-methyl -4- hydroxyl pyrrolidine -2- carboxylate] (6)
Oxalyl chloride (24.5mL, 35.6g, 281mmol) is added to paranitrobenzoic acid 5 (43g, 92.3mmol) and MDF (6mL) is in the stirred suspension of anhydrous DCM (600mL).After initial effervescence, reaction suspension becomes solution, and allows It stirs mixture 16 hours at room temperature.By determining the conversion ratio of acid chloride with the sample of MeOH reaction mixture, and And double-the methyl ester observed by LC/MS.Most of solvent is removed by evaporating under reduced pressure；Obtained concentration is molten Liquid is re-dissolved in the anhydrous DCM of minimum, and is beaten with diethyl ether.The yellow mercury oxide being collected by filtration is used Cold diethyl ether washing, and it is 1 hour dry in 40 DEG C of vacuum drying ovens.Within the 25min period, -40 DEG C (dry ice/ CH3CN solid acid chloride is added batch-wise to (2S, 4R)-methyl -4- hydroxyl pyrrolidine -2- carboxylate salts acidulants under) (38.1g, 210mmol) and TEA (64.5mL, g, 463mmol) are in the stirred suspension in DCM (400mL).At once, pass through LC/MS (2.47min (ES+) m/z (relative intensity 721 ([M+H]+., 100) and determine that reaction is completed.Simultaneously with DCM (200mL) dilution And with 1N HCl (300mL), saturation NaHCO3(300mL), salt water (400mL), dry (MgSO4) washing mixture, filtering is simultaneously Solvent is evaporated, in vacuo to obtain the pure products 6 (66.7g, 100%) for orange solids.[α]22 D=-46.1 ° of (c= 0.47, CHCl3)；1H NMR (400MHz, CDCl3) (rotational isomer) δ 7.63 (s, 2H), 6.82 (s, 2H), 4.79-4.72 (m, 2H), 4.49-4.28 (m, 6H), 3.96 (s, 6H), 3.79 (s, 6H), 3.46-3.38 (m, 2H), 3.02 (d, 2H, J= 11.1Hz), 2.48-2.30 (m, 4H), 2.29-2.04 (m, 4H)；13C NMR (100MHz, CDCl3) (rotational isomer) δ 172.4,166.7,154.6,148.4,137.2,127.0,109.7,108.2,69.7,65.1,57.4,57.0,56.7, 52.4,37.8,29.0；IR (ATR, CHCl3) 3410 (br), 3010,2953,1741,1622,1577,1519,1455,1429, 1334,1274,1211,1177,1072,1050,1008,871cm-1；MS(ES+) m/z (relative intensity) 721 ([M+H]+., 47), 388(80)；HRMS[M+H]+.Theoretical C31H36N4O16M/z721.2199 has found (ES+)m/z 721.2227。
(e) 1,1 '-[[(propane -1,3- diyl) two oxygroups] bis- methoxyl group -1,2,3 (11aS, 2R) -2- (hydroxyl) -7-, 10,11,11a- hexahydro -5H- pyrrolo- [2,1-c] [1,4]-benzodiazepine5,11- diketone] (7)
Method A: solution of the nitro ester 6 (44g, 61.1mmol) in MeOH (2.8L) is added to 3 neck round-bottom flask of 5L In newly buyNickel (~50g, in water~50% slurry) and uprising boiling particle in.Heating mixing under reflux Then object is handled dropwise with the solution of hydrazine hydrate (21.6mL, 22.2g, 693mmol) in MeOH (200mL), at this point, observation To sufficient effervesce.It is careful to add in addition when completing to add (~45min)Nickel stops until effervesce, and arranges Initial yellow reaction mixture out.Mixture 5min is in addition heated under reflux, at this point, by TLC (90:10v/v CHCl3/ ) and LC/MS (2.12min (ES+) m/z (relative intensity) 597 ([M+H] MeOH+., 100) reaction be considered as completion.Pass through packet immediately The sinter funnel heat filtering reaction mixture of containing diatomite (celite) and vaccum suction pipe.Reduce filtrate body by being evaporated in vacuo Product, at this point, forming colourless precipitate, precipitating is collected by filtration and drying in vacuum desiccator to provide 7 (31g, 85%). [α]27 D=+404 ° (c=0.10, DMF)；1H NMR (400MHz, DMSO-d6) δ 10.2 (s, 2H, NH), 7.26 (s, 2H), 6.73 (s, 2H), 5.11 (d, 2H, J=3.98Hz, OH), 4.32-4.27 (m, 2H), 4.19-4.07 (m, 6H), 3.78 (s, 6H), 3.62 (dd, 2H, J=12.1,3.60Hz), 3.43 (dd, 2H, J=12.0,4.72Hz), 2.67-2.57 (m, 2H), 2.26 (p, 2H, J=5.90Hz), 1.99-1.89 (m, 2H)；13C NMR (100MHz, DMSO-d6) δ 169.1,164.0,149.9,144.5, 129.8,117.1,111.3,104.5,54.8,54.4,53.1,33.5,27.5；IR (ATR, pure) 3438,1680,1654, 1610,1605,1516,1490,1434,1379,1263,1234,1216,1177,1156,1115,1089,1038,1018, 952,870cm-1；MS(ES+) m/z (relative intensity) 619 ([M+Na]+., 10), 597 ([M+H]+., 52), 445 (12), 326 (11)；HRMS[M+H]+.Theoretical C29H32N4O10M/z 597.2191 has found (ES+)m/z597.2205。
Method B: by suspension of the 10%Pd/C (7.5g, 10%w/w) in DMF (40mL) be added to nitro ester 6 (75g, 104mmol) in the solution in DMF (360mL).It is more than 8 hours that the suspension is hydrogenated in Parr hydrogenation apparatus.Hydrogen absorption stops After only, reaction process is monitored by LC/MS.Solid Pd/C is removed by filtration, and by under vacuum (being lower than 10mbar) Filtrate is concentrated in 40 DEG C of rotary evaporations, to provide comprising trace DMF and remain the dark oil of charcoal.At 40 DEG C, in water-bath, (rotation is steamed Hair bath) on residue absorbed into (digestion, digest) in EtOH (500mL), and the suspension being obtained by filtration by diatomite It liquid and is washed with ethyl alcohol (500mL), to generate limpid filtrate.Hydrazine hydrate (10mL, 321mmol) is added in solution And the reaction mixture is heated under reflux.It after 20 minutes, observes that white precipitate is formed, and allows to flow back and continue in addition 30 minutes.Allow for mixture to be cooled to room temperature, and precipitating is recovered by filtration, with diethyl ether (the 2:1 volume ratio of precipitating) Washing and the drying in vacuum desiccator, to provide 7 (50g, 81%).The analysis data of method B: the analysis obtained with method A Data it is identical (optical activity,1H NMR, LC/MS and TLC).
(f) 1,1 '-[[(propane -1,3- diyl) two oxygroups] bis- (11aS, 2R) -2- (t-butyldimethylsilyloxies Base) -7- methoxyl group -1,2,3,10,11,11a- hexahydro -5H- pyrrolo- [2,1-c] [1,4]-benzodiazepine- 5,11- two Ketone] (8)
TBSCl (27.6g, 182.9mmol) and imidazoles (29.9g, 438.8mmol) are added under 0 DEG C (ice/acetone) Four lactams 7 (21.8g, 36.6mmol) are in the turbid solution in anhydrous DMF (400mL).Allow to stir in a nitrogen atmosphere Mixture 3 hours, which passed through LC/MS (3.90min (ES+) m/z (relative intensity) 825 ([M+H] later+., 100) and judgement Confirmation reaction is completed.Reaction mixture is poured on ice (~1.75L), and allows it to be warming up to room temperature under stiring.Pass through The white precipitate that vacuum filter is collected, uses H2O, diethyl ether washs, and dry in vacuum desiccator, pure to provide 8 (30.1g, 99%).[α]23 D=+234 ° of (c=0.41, CHCl3)；1H NMR (400MHz, CDCl3) δ 8.65 (s, 2H, NH), 7.44 (s, 2H), 6.54 (s, 2H), 4.50 (p, 2H, J=5.38Hz), 4.21-4.10 (m, 6H), 3.87 (s, 6H), 3.73- 3.63 (m, 4H), 2.85-2.79 (m, 2H), 2.36-2.29 (m, 2H), 2.07-1.99 (m, 2H), 0.86 (s, 18H), 0.08 (s, 12H)；13C NMR (100MHz, CDCl3) δ 170.4,165.7,151.4,146.6,129.7,118.9,112.8,105.3, 69.2,65.4,56.3,55.7,54.2,35.2,28.7,25.7,18.0, -4.82 and -4.86；IR (ATR, CHCl3) 3235, 2955,2926,2855,1698,1695,1603,1518,1491,1446,1380,1356,1251,1220,1120,1099, 1033cm-1；MS(ES+) m/z (relative intensity) 825 ([M+H]+ ., 62), 721 (14), 440 (38)；HRMS[M+H]+.It is theoretical C41H60N4O10Si2M/z 825.3921 has found (ES+)m/z825.3948。
(g) 1,1 '-[[(propane -1,3- diyl) two oxygroups] bis- [(11aS, 2R) -2- (t-butyldimethylsilyloxies Base) -7- methoxyl group -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -1,2,3,10,11,11a- hexahydro -5H- pyrroles And [2,1-c] [1,4]-benzodiazepine5,11- diketone] (9)
Under -30 DEG C (dry ice/ethylene glycol), in a nitrogen atmosphere by n-BuLi (the 1.6M solution of the 68.3mL in hexane, It 109mmol) is added dropwise to four lactams 8 (30.08g, 36.4mmol) in the stirred suspension in anhydrous THF (600mL). Reaction mixture is stirred for 1 hours at such a temperature (being reddish orange now) for permission, at this point, SEMCl is added dropwise The solution of (19.3mL, 18.2g, 109mmol) in anhydrous THF (120mL).Reaction mixture is allowed to be to slowly warm up to room Temperature, and stir 16 hours in a nitrogen atmosphere.Pass through TLC (EtOAc) and LC/MS (4.77min (ES+) m/z (relative intensity) 1085([M+H]+., 100) and judgement, it reacts and is considered as completion.THF is removed by evaporating under vacuum, and obtained residual is dissolved In EtOAc (750mL), H is used2O (250mL), salt water (250mL) washing, dry (MgSO4), filtered under vacuum and evaporation, with It is provided as four lactams, the 9 (max of the thick N10-SEM- protection of oilym39.5g, 100%).Make product carry out next step and Without purifying.[α]23 D=+163 ° of (c=0.41, CHCl3)；1H NMR (400MHz, CDCl3) δ 7.33 (s, 2H), 7.22 (s, 2H), 5.47 (d, 2H, J=9.98Hz), 4.68 (d, 2H, J=9.99Hz), 4.57 (p, 2H, J=5.77Hz), 4.29-4.19 (m, 6H), 3.89 (s, 6H), 3.79-3.51 (m, 8H), 2.87-2.81 (m, 2H), 2.41 (p, 2H, J=5.81Hz), 2.03- 1.90 (m, 2H), 1.02-0.81 (m, 22H), 0.09 (s, 12H), 0.01 (s, 18H)；13C NMR (100MHz, CDCl3)δ 170.0,165.7,151.2,147.5,133.8,121.8,111.6,106.9,78.1,69.6,67.1,65.5,56.6, 56.3,53.7,35.6,30.0,25.8,18.4,18.1, -1.24, -4.73；IR (ATR, CHCl3) 2951,1685,1640, 1606,1517,1462,1433,1360,1247,1127,1065cm-1；MS(ES+) m/z (relative intensity) 1113 ([M+Na]+ ., 48), 1085 ([M+H]+., 100) and 1009 (5), 813 (6)；HRMS[M+H]+.Theoretical C53H88N4O12Si4M/z 1085.5548, hair Existing (ES+)m/z 1085.5542。
(h) 1,1 '-[[(propane -1,3- diyl) two oxygroups] bis- [(11aS, 2R) -2- hydroxyl -7- methoxyl group -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -1,2,3,10,11,11a- hexahydro -5H- pyrrolo- [2,1-c] [1,4] benzo two Azepine5,11- diketone]] (10)
TBAF (150mL, 1.0M solution in THF, 150mmol) is added to thick double-silyl ester 9 at room temperature [84.0g(maxm56.8g), 52.4mmol] in the solution of the stirring in THF (800mL).After stirring 1 hour, pass through TLC (5:5v/v CHCl3/ MeOH) analysis reaction mixture display reaction completion.At room temperature, it is removed under reduced pressure by evaporation THF, and obtained residual is dissolved in EtOAc (500mL) and uses NH4Cl (300mL) washing.It is washed with salt water (60mL) It washs, dry (MgSO4), filtering and the organic layer for evaporating combination under reduced pressure, to provide crude product.It is pure by flash chromatography Change (gradient eluent: 100%CHCl3To 96:4v/v CHCl3/ MeOH) it is produced as the four pure lactams 10 of white foam (36.0g, 79%).LC/MS3.33min (ES+) m/z (relative intensity) 879 ([M+Na]+., 100), 857 ([M+H]+., 40)； [α]23 D=+202 ° of (c=0.34, CHCl3)；1H NMR (400MHz, CDCl3) δ 7.28 (s, 2H), 7.20 (s, 2H), 5.44 (d, 2H, J=10.0Hz), 4.72 (d, 2H, J=10.0Hz), 4.61-4.58 (m, 2H), 4.25 (t, 4H, J=5.83Hz), 4.20- 4.16 (m, 2H), 3.91-3.85 (m, 8H), 3.77-3.54 (m, 6H), 3.01 (br s, 2H, OH), 2.96-2.90 (m, 2H), 2.38 (p, 2H, J=5.77Hz), 2.11-2.05 (m, 2H), 1.00-0.91 (m, 4H), 0.00 (s, 18H)；13C NMR (100MHz, CDCl3) δ 169.5,165.9,151.3,147.4,133.7,121.5,111.6,106.9,79.4,69.3, 67.2,65.2,56.5,56.2,54.1,35.2,29.1,18.4, -1.23；IR (ATR, CHCl3) 2956,1684,1625, 1604,1518,1464,1434,1361,1238,1058,1021cm-1；MS(ES+) m/z (relative intensity) 885 ([M+29]+., 70), 857 ([M+H]+., 100), 711 (8), 448 (17)；HRMS[M+H]+.Theoretical C41H60N4O12Si2M/z 857.3819, hair Existing (ES+)m/z 857.3826。
(i) 1,1 '-[[(propane -1,3- diyl) two oxygroups] bis- [(11aS) -7- methoxyl group -2- oxo -10- ((2- (three Methyl silicane base) ethyoxyl) methyl) -1,2,3,10,11,11a- hexahydro -5H- pyrrolo- [2,1-c] [1,4] benzodiazepine * It is miscellaneous5,11- diketone]] (11)
At Ar, by glycol 10 (25.6g, 30mmol, 1 equivalent), NaOAc (6.9g, 84mmol, 2.8 equivalent) and TEMPO (188mg, 1.2mmol, 0.04 equivalent) is dissolved in DCM (326mL).- 8 DEG C (internal temperatures) are cooled to, and 15 By batch addition TCCA (9.7g, 42mmol, 1.4 equivalent) in minute.After 30 minutes, TLC (EtOAc) and LC/MS [3.60min. (ES+) m/z (relative intensity) 854.21 ([M+H]+., 40), (ES-) m/z (relative intensity) 887.07 ([M-H+Cl]-., 10)] Indicator Reaction is completed.It adds cold DCM (200mL), and in sodium bicarbonate/hypo solution (1:1v/v with saturation； 200mL × 2) washing before, pass through diatomite filter plates mixture.Use MgSO4Organic layer is dried, filtered, and vacuum is removed Solvent is removed, to generate yellow/orange sponge (25.4g, 99%).LC/MS [3.60min. (ES+) m/z (relative intensity) 854.21([M+H]+., 40)；[α]20 D=+291 ° of (c=0.26, CHCl3)；1H NMR (400MHz, CDCl3) δ 7.32 (s, 2H), 7.25 (s, 2H), 5.50 (d, 2H, J=10.1z), 4.75 (d, 2H, J=10.1Hz), 4.60 (dd, 2H, J=9.85, 3.07Hz), 4.31-4.18 (m, 6H), 3.89-3.84 (m, 8H), 3.78-3.62 (m, 4H), 3.55 (dd, 2H, J=19.2, 2.85Hz), 2.76 (dd, 2H, J=19.2,9.90Hz), 2.42 (p, 2H, J=5.77z), 0.98-0.91 (m, 4H), 0.00 (s, 18H)；13C NMR (100MHz, CDCl3) δ 206.8,168.8,165.9,151.8,148.0,133.9,120.9,111.6, 107.2,78.2,67.3,65.6,56.3,54.9,52.4,37.4,29.0,18.4, -1.24；IR (ATR, CHCl3) 2957, 1763,1685,1644,1606,1516,1457,1434,1360,1247,1209,1098,1066,1023cm-1；MS(ES+)m/ Z (relative intensity) 881 ([M+29]+., 38), 853 ([M+H]+., 100), 707 (8), 542 (12)；HRMS[M+H]+.It is theoretical C41H56N4O12Si2M/z 853.3506 has found (ES+)m/z 853.3502。
(j) 1,1 '-[[(propane -1,3- diyl) two oxygroups] bis- (11aS) -7- methoxyl group -2- [[(trifluoromethyl) sulphonyl Base] oxygroup] -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -1,10,11,11a- tetrahydro -5H- pyrrolo- [2,1- C] [1,4]-benzodiazepine5,11- diketone (12)
Under -45 DEG C (dry ice/acetonitrile), in a nitrogen atmosphere, with 1 part by anhydrous 2,6- lutidines (5.15mL, 4.74g, 44.2mmol) injection diketone 11 (6.08g, 7.1mmol) being vigorously stirred in solution in anhydrous DCM (180mL).Fastly Speed injects the anhydrous trifluoromethanesulfanhydride anhydride (7.2mL, 12.08g, 42.8mmol) taken out from the bottle of fresh opening dropwise, together When maintain the temperature at -40 DEG C or less.Allow that reaction mixture is stirred for 1 hours at -45 DEG C, at this point, TLC (50/50v/v just oneself Alkane/EtOAc) show that starting material is completely depleted.Cold reaction mixture is diluted with DCM (200mL) immediately, and is acutely shaken It shakes, with water (1 × 100mL), 5% citric acid solution (1 × 200mL), the NaHCO being saturated3(200mL), salt water (100mL) are washed It washs, and dry (MgSO4).Under reduced pressure filtering and evaporation provide crude product, by flash column chromatography (gradient elution: 90: 10v/v n-hexane/EtOAc to 70:30v/v n-hexane/EtOAc) crude product is purified to be provided as the diene alcohol of yellow colored foam Triflate (salt) (5.5g, 70%).LC/MS 4.32min (ES+) m/z (relative intensity) 1139 ([M+Na]+., 20)； [α]24 D=+271 ° of (c=0.18, CHCl3)；1H NMR (400MHz, CDCl3) δ 7.33 (s, 2H), 7.26 (s, 2H), 7.14 (t, 2H, J=1.97Hz), 5.51 (d, 2H, J=10.1Hz), 4.76 (d, 2H, J=10.1Hz), 4.62 (dd, 2H, J=11.0, 3.69Hz), 4.32-4.23 (m, 4H), 3.94-3.90 (m, 8H), 3.81-3.64 (m, 4H), 3.16 (ddd, 2H, J=16.3, 11.0,2.36Hz), 2.43 (p, 2H, J=5.85Hz), 1.23-0.92 (m, 4H), 0.02 (s, 18H)；13C NMR (100MHz, CDCl3) δ 167.1,162.7,151.9,148.0,138.4,133.6,120.2,118.8,111.9,107.4,78.6,67.5, 65.6,56.7,56.3,30.8,29.0,18.4, -1.25；IR (ATR, CHCl3) 2958,1690,1646,1605,1517, 1456,1428,1360,1327,1207,1136,1096,1060,1022,938,913cm-1；MS(ES+) m/z (relative intensity 1144([M+28]+., 100), 1117 ([M+H]+., 48), 1041 (40), 578 (8)；HRMS[M+H]+.It is theoretical C43H54N4O16Si2S2F6M/z 1117.2491 has found (ES+)m/z 1117.2465。
Embodiment 1
(a) (S)-trifluoromethanesulfonic acid 8- (3- (((S) -2- (4- aminophenyl) -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1, 4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) second Oxygroup) methyl) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- base ester (13)
By Pd (PPh3)4(116.9mg, 0.101mmol) be added to double-enol triflate 12 (5.65g, 5.06mmol), 4- aminophenyl boronic acid pinacol ester (1g, 4.56mmol), Na2CO3(2.46g, 23.2mmol), MeOH In the stirring mixture of (37mL), toluene (74mL) and water (37mL).Permission is stirred to react mixing at 30 DEG C in a nitrogen atmosphere Object 24 hours, after the time, all borates were depleted.Then before taking out residual from EtOAc (150mL), evaporation Reaction mixture uses H to drying2O (2 × 100mL), salt water (150mL) washing, dry (MgSO4), it filters under reduced pressure And evaporation, to provide crude product.Pass through purified by flash chromatography (gradient elution: 80:20v/v hexane/EtOAc to 60:40v/v Hexane/EtOAc) provide product 13 (2.4g, 45%) for weak yellow foam.LC/MS 4.02min (ES+) m/z is (relatively strong Degree) 1060.21 ([M+H]+., 100)；1H-NMR:(CDCl3, 400MHz) δ 7.40 (s, 1H), 7.33 (s, 1H), 7.27 (bs, 3H), 7.24 (d, 2H, J=8.5Hz), 7.15 (t, 1H, J=2.0Hz), 6.66 (d, 2H, J=8.5Hz), 5.52 (d, 2H, J= 10.0Hz), 4.77 (d, 1H, J=10.0Hz), 4.76 (d, 1H, J=10.0Hz), 4.62 (dd, 1H, J=3.7,11.0Hz), 4.58 (dd, 1H, J=3.4,10.6Hz), 4.29 (t, 4H, J=5.6Hz), 4.00-3.85 (m, 8H), 3.80-3.60 (m, 4H), 3.16 (ddd, 1H, J=2.4,11.0,16.3Hz), 3.11 (ddd, 1H, J=2.2,10.5,16.1Hz), 2.43 (p, 2H, J=5.9Hz), 1.1-0.9 (m, 4H), 0.2 (s, 18H).13C-NMR:(CDCl3, 100MHz) and δ 169.8,168.3, 164.0,162.7,153.3,152.6,149.28,149.0,147.6,139.6,134.8,134.5,127.9,127.5, 125.1,123.21,121.5,120.5,120.1,116.4,113.2,108.7,79.8,79.6,68.7,68.5,67.0, 66.8,58.8,58.0,57.6,32.8,32.0,30.3,19.7,0.25.
(b) (S) -2- (4- aminophenyl) -8- (3- (((S) -2- cyclopropyl -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1, 4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -1H- Benzo [e] pyrrolo- [1,2-a] [1,4] diaza5,11 (10H, 11aH)-diketone (14)
Under an argon atmosphere, by triphenylarsine (0.24g, 0.8mmol), silver oxide (I) (1.02g, 4.4mmol), cyclopropyl Ylboronic acid (0.47g, 5.5mmol) and starting material 13 (1.15g, 1.1mmol) are dissolved in dioxane (30mL).With pestle and Mortar grinder tripotassium phosphate (2.8g, 13.2mmol), and be quickly added in reaction mixture.Reaction mixture is emptied, And with argon cleaning 3 times, and it is heated to 71 DEG C.Bis- (chlorination benzonitrile) palladiums (II) (84mg, 0.22mmol) are added, and are emptied Reaction vessel and with argon cleaning 3 times.After ten minutes, extract a small amount of sample for by TLC (80:20v/v ethyl acetate/oneself Alkane) and LC/MS analysis.After 30 minutes, reaction carries out (LC/MS analysis instruction starting material is totally consumed) completely, Yi Jitong The filter plates reactant crossing diatomite and being washed with ethyl acetate (400mL).With water (2x200mL) and salt water (2x200mL) Wash filtrate.Use MgSO4Dry organic layer, vacuum filter remove solvent.Pass through silica gel column chromatography (30:70v/v hexane/acetic acid Ethyl ester) purify the product 14 (0.66g, 63%) provided as orange/yellow solid.Method 1, LC/MS (3.85min (ES+)m/ Z (relative intensity) 952.17 ([M+H]+, 100).1H NMR (400MHz, CDCl3) δ 7.36 (d, 2H, J=8.4Hz), 7.30 (s, 1H), 7.25-7.19 (m, 4H), 6.68 (s, 1H), 6.62 (d, 2H, J=8.4Hz), 5.49 (dd, 2H, J=5.6,10.0Hz), 4.73 (app.t, 2H, J=10.8Hz), 4.54 (dd, 1H, J=3.2,10.4Hz), 4.40 (dd, 1H, J=3.2,10.4Hz), 4.29-4.23 (m, 4H), 3.91-3.85 (m, 7H), 3.80-3.71 (m, 2H), 3.70-3.61 (m, 2H), 3.38-3.32 (m, 1H), 3.12-3.01 (m, 1H), 2.50-2.69 (m, 1H), 2.40 (q, 2H, J=5.6Hz), 1.50-1.43 (m, 1H), 0.99- 0.71 (m, 6H), 0.54-0.59 (m, 2H), 0.00 (s, 18H) ppm.
(c) (S) -2- (4- aminophenyl) -8- (3- (((S) -2- cyclopropyl -7- methoxyl group -5- oxo -5,11a- dihydro - 1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -1H- benzo [e] pyrrole Cough up simultaneously [1,2-a] [1,4] diaza5 (11aH) -one (15)
Under an argon atmosphere, the bis- lactams 14 (0.66g, 0.69mmol) of SEM are dissolved in THF (23mL) and are cooled down To -78 DEG C.It is added dropwise in 5 minutesSolution (1M in 1.7mL, THF) while monitoring temperature.20 points Zhong Hou extracts a small amount of sample, and is washed with water and analyzes for LC/MS.Addition water (50mL) simultaneously removes cryostat.It extracts organic Layer, and washed with salt water (60mL).Use CH2Cl2The water layer that/MeOH (90/10v/v) (2 × 50mL) washing combines.Use MgSO4It is dry The organic layer of constipation conjunction, filtering, and solvent is removed in vacuum.Crude product is dissolved in MeOH (48mL), CH2Cl2(18mL) and water In (6mL), and enough silica gel is added to form dense suspension.After stirring 5 days, is filtered and suspended by sinter funnel Liquid, and use CH2Cl2/ MeOH (9:1) (~200mL) washing is eluted completely until product.It has been washed with salt water (2x70mL) Machine layer, uses MgSO4Dry, vacuum filter removes solvent.Pass through silica gel chromatography (100%CHCl3To 96:4v/v CHCl3/ MeOH) provide product 15 (302mg, 66%) for yellow solid.Method 1, LC/MS (2.42min (ES+) m/z (phase To intensity) 660.74 ([M+H]+, 30).1H NMR (400MHz, CDCl3) δ 7.86 (d, 1H, J=3.6Hz), 7.78 (d, 1H, J =3.6Hz), 7.58-7.44 (m, 3H), 7.34-7.20 (m, 3H), 6.88-6.66 (m, 4H), 4.35-4.15 (m, 6H), 3.95-3.75 (m, 7H), 3.39-3.22 (m, 1H), 3.14-3.04 (m, 1H), 2.93-2.85 (m, 1H), 2.46-2.36 (m, 2H), 1.49-1.41 (m, 1H), 0.80-0.72 (m, 2H), 0.58-0.51 (app.s, 2H) ppm.
(d) ((2S) -1- (((2S) -1- ((4- (8- (3- ((2- cyclopropyl -7- methoxyl group -5- oxo -5,11a- dihydro - 1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -5- oxo -5,11a- Dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- oxopropan -2- base) ammonia Base)-3- methyl-1-oxo-butanes-2- base) allyl carbamate (16)
In the degassing round-bottomed flask filled with argon gas, by HO-Ala-Val-alloc (149.6mg, 0.549mmol) and EEDQ (135.8mg, 0.549mmol) is dissolved in dry CH2Cl2In the 9:1 mixture of/MeOH (5mL).It is wrapped up and being burnt with aluminium foil Bottle, and before adding starting material 15 (302mg, 0.457mmol), allow to be stirred at room temperature reaction mixture 1 hour. Before removing volatile matter by rotary evaporation under reduced pressure, reaction mixture other 40 hours are stirred at room temperature (after reaction It is LC/MS, RT starting material 2.32min, (ES+660.29([M+H]+., 100)).Pass through silica gel chromatographic column (100%CHCl3Extremely 90/10v/v CHCl3/ MeOH) direct purification crude product, pure products (16) are provided with 42% yield (174mg).Method 2, LC/ MS (2.70min (ES+) m/z (relative intensity) 914.73 ([M+H]+, 60), 660.43 (60), 184.31 (100).
(e) (2S) -2- amino-N- ((2S) -1- ((4- (8- (3- ((2- cyclopropyl -7- methoxyl group -5- oxo -5,11a- Dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -5- oxo - 5,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- oxopropan -2- Base) -3- methylbutyryl amine (17)
In the round-bottomed flask filled with argon gas, before adding pyrrolidines (41 μ L, 0.21mmol), by starting material 16 (170mg, 0.185mmol) is dissolved in dry CH2Cl2In (5mL).In addition Pd (PPh3)4(14mg, 0.084mmol) it Before, flask is purged/refilled three times with argon gas, and repeat flushing operation.After 1 hour, observe that starting material is consumed completely To the greatest extent (reaction after be LC/MS), and by Et2O (50mL), which is added to reaction mixture, to be allowed to stir until product is analysed from solution Out.Filter solid is crossed by sinter funnel, and uses Et2O washes twice (2x25mL).Flask, and consolidating separation are collected in displacement Body is dissolved in CHCl3In (100mL or until all products all pass through sinter funnel).Then it is removed under reduced pressure by rotary evaporation Volatile matter is removed to provide crude product 17, (168mg) will be used directly in a subsequent step.LC/MS method 2 (2.70min (ES+) m/z (relative intensity) 830.27 ([M+H]+, 50), 660.13 (80), 171.15 (100)).
(f) N- ((R) -1- (((S) -1- ((4- ((S) -8- (3- ((oxo -5 (S) -2- cyclopropyl -7- methoxyl group -5-, 11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -5- oxygen Generation -5,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- oxo third Alkane-2- base) amino)-3- methyl-1-oxo-butanes-2- base)-1- (3- (2,5- dioxo-2,5- dihydro-1H- pyrroles-1- base) Third amino) eight oxa- heptacosane -27- amide (18) of -3,6,9,12,15,18,21,24-.
In the round-bottomed flask for being purged and being filled with argon gas, by starting material 17 (154mg, 0.185mmol) and EDCI.HCl (110mg, 0.185mmol) is dissolved in dry CH2Cl2In (5mL).In addition PEG8Maleimide Before (35.6mg, 0.185mmol), mixture is stirred at room temperature 1 hour, and is stirred to react mixture other 16 hours (or until reaction completion, being monitored by LC/MS).Use CH2Cl2(50mL) diluting reaction solution, and with MgSO4Before drying Use H2O (50mL) and salt water (50mL) wash organic matter, filter and remove solvent by rotary evaporation under reduced pressure, thick to provide Product.Silica gel column chromatography (100%CHCl3To 85/15v/v CHCl3/ MeOH) the desired product (135mg) of purifying generation, still The unreacted PEG that (passing through LC/MS, 2.21min, method 2) has observed trace remaining8Maleimide.Automatic reverse phase silica gel Chromatography (H2O/CH3CN) be successfully removed impurity (referring to the general information of condition), provide pure final product (18, starting In the 37mg pure products of 110mg, 33%).Overall productivity=17%.Method 2, (2.58min (ES+) m/z is (relatively strong by LC/MS Degree) 1404.03 ([M+H]+, 20), 702.63 (100)).1H NMR (400MHz, CDCl3) δ 7.91 (t, J=3.5Hz, 1H), 7.80 (d, J=4.0Hz, 1H), 7.75 (d, J=8.8Hz, 1H), 7.69 (d, J=8.7Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m, 2H), 6.87 (d, J=10.5Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J=10.2,5.2Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J=16.1,4.1Hz, 1H), 2.62-2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J=9.2,5.3Hz, 2H), 0.57 (dd, J=9.2,5.3Hz, 2H) are not observed NH。
Embodiment 2
(a) (R) -2- ((R) -2- ((((9H- fluorenes -9- base) methoxyl group) carbonyl) amino) -3- methylbutylamino) propionic acid (20b)
By HO-Ala-Val-H 20a (350mg, 1.86mmol) and Na2CO3(493mg, 4.65mmol) is dissolved in distillation H2In O (15mL), and before addition dioxane (15mL) mixture is cooled to 0 DEG C (part that amino-acid salt occurs is heavy It forms sediment).With vigorous stirring, through 10 minutes, Fmoc-Cl (504mg, 1.95mmol) is added dropwise in dioxane (15mL) Solution.Before removing ice bath, 0 DEG C the mixture stirred to get 2 hours, then persistently stir 16 hours.Lead under reduced pressure Rotation evaporation of solvent is crossed, and residue is dissolved in water (150mL).PH is adjusted to 2 from 9 with 1N HCl, is then used EtOAc (3x100mL) aqueous layer extracted.The organic matter merged is washed with salt water (100mL), uses MgSO4It dries, filters and is depressurizing Volatile matter is removed to provide pure HO-Ala-Val-Fmoc 20b (746mg, 97% yield) by rotary evaporation down.LC/ MS2.85min (ES+) m/z (relative intensity) 410.60；1H-NMR (400MHz, CDCl3) δ 7.79 (d, J=7.77Hz, 2H), 7.60 (d, J=7.77Hz, 2H), 7.43 (d, J=7.5Hz, 2H), 7.34 (d, J=7.5Hz, 2H), 6.30 (bs, 1H), 5.30 (bs, 1H), 4.71-7.56 (m, 1H), 4.54-4.36 (m, 2H), 4.08-3.91 (m, 1H), 2.21-2.07 (m, 1H), 1.50 (d, J=7.1Hz, 3H), 1.06-0.90 (m, 6H).
(b) ((S)-3- methyl-1-oxo-1- (((S)-1- oxo-1- ((4- (4,4,5,5- tetramethyl-1,3,2- dioxy Miscellaneous borine -2- base) phenyl) amino) propane -2- base) amino) butane -2- base) carbamic acid (9H- fluorenes -9- base) methyl ester ((9H-fluoren-9-yl)methyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)propan-2-yl)amino)butan-2- yl)carbamate)(20)
At room temperature, 4- aminophenyl boronic acid pinacol ester (146.9mg, 0.67mmol) is added to and has previously stirred 30 Minute the flask with argon cleaning in HO-Ala-Val-Fmoc 20b (330mg, 0.8mmol), DCC (166mg, 0.8mmol) and DMAP (5mg, catalyst) is in the solution in anhydrous DCM (8mL).Then it is mixed to allow to be stirred at room temperature reaction Object is closed to stay overnight.Along with LCMS and TLC after reaction.Use CH2Cl2(50mL) diluted reaction mixture, and with MgSO4Drying Before use H2O and salt water washing organic matter filter under reduced pressure and remove solvent by rotary evaporation.Crude product drying is supported on On silica gel chromatographic column (hexane/EtOAc, 6:4), and it is separated into 88% yield (360mg) pure products 20 of white solid.
(c) trifluoromethanesulfonic acid 8- (3- ((2- (4- ((S) -2- ((S) -2- ((((9H- fluorenes -9- base) methoxyl group) carbonyl) ammonia Base) -3- methylbutylamino) the third amino) phenyl) -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) second Oxygroup) methyl) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) third Oxygroup) -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- four Hydrogen -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- base ester (21)
It will double-triflate 12 (2.03g, 1.81mmol), pinacol borate (1g, 1.63mmol) and Na2CO3 (881mg, 8.31mmol) is dissolved in toluene/MeOH/H2In the mixture of O (2:1:1,40mL).Adding four (triphenylphosphines) Before palladium (0) (41mg, 0.035mmol), is purged with argon gas and fill reaction flask, and extremely by reaction mixture heated overnight 30℃.Solvent is removed under reduced pressure, and residual is extracted in H2It is extracted in O (100mL) and with EtOAc (3x100mL).With Salt water (100mL) washs combined organic matter, uses MgSO4It dries, filters and passes through rotary evaporation under reduced pressure and remove volatile matter. By silica gel chromatographic column (hexane/EtOAc, 8:2to 25:75) purification of crude product to provide pure the 21 of 33% yield (885mg). LC/MS 3.85min (ES+) m/z (relative intensity) 1452.90；1H NMR (400MHz, CDCl3) δ 7.78-7.16 (m, 17H), 7.13 (s, 1H), 6.51-6.24 (m, 1H), 5.51 (dd, J=10.0,5.1Hz, 2H), 5.36-5.11 (m, 1H), 4.74 (dd, J=10.1,4.4Hz, 2H), 4.70-4.53 (m, 2H), 4.47 (d, J=6.4Hz, 1H), 4.37 (d, J=7.2Hz, 1H), 4.27 (m, 4H), 4.20-4.14 (m, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.77 (ddd, J=16.7,9.0,6.4Hz, 3H), 3.71-3.61 (m, 2H), 3.24-2.91 (m, 3H), 2.55-2.33 (m, 2H), 2.22-2.07 (m, 1H), 1.52-1.37 (m, 3H), 1.04-0.86 (m, 10H), 0.00 (s, 18H).
(d) ((2S) -1- (((2S) -1- ((4- (8- (3- ((2- cyclopropyl -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] two Azepine8- yl) oxygroup) propoxyl group) -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) Methyl -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- Oxopropan-2- base) amino)-3- methyl-1-oxo-butanes-2- base) carbamic acid (9H- fluorenes-9- base) methyl ester (22) ((9H-fluoren-9-yl)methyl((2S)-1-(((2S)-1-((4-(8-(3-((2-cyclopropyl-7-methoxy- 5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H- benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-7-methoxy-5,11-dioxo- 10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e] pyrrolo[1,2-a][1,4]diazepin-2-yl)phenyl)amino)-1-oxopropan-2-yl)amino)-3- methyl-1-oxobutan-2-yl)carbamate)
Under an argon atmosphere, triphenylarsine (42mg, 0.137mmol) is added to PBD- triflate 21 (250mg, 0.172mmol), cyclopropylboronic acid (73.9mg, 0.86mmol), silver oxide (159mg, 0.688mmol) and tricresyl phosphate Potassium (438mg, 2.06mmol) is in the mixture in dry dioxane (10mL).It is reacted 3 times, and added double with argon cleaning (benzonitrile) palladium chloride (II) (13.2mg, 0.034mmol).Before it will react and be warming up to 75 DEG C, reacted 3 times with argon cleaning More than, and stirring 10 minutes.By diatomite filter plates reaction mixture, then rinsed with ethyl acetate.Under reduced pressure Solvent is removed by rotary evaporation.The residual made carries out flash column chromatography (silica gel；1% methanol/chloroform).It collects and merges Pure part, and excessive eluent is removed to provide desired product 22 (132mg, 50% production by rotary evaporation under reduced pressure Rate).LC/MS3.83min (ES+) m/z (relative intensity) 1345.91；1H NMR (400MHz, CDCl3) δ 7.88-7.14 (m, 17H), 6.69 (s, 1H), 6.45-6.25 (m, 1H), 5.57-5.41 (m, 2H), 5.34-5.14 (m, 1H), 4.78-4.67 (m, 2H), 4.62-4.55 (m, 1H), 4.50-4.45 (m, 2H), 4.51-4.44 (m, 1H), 4.31-4.21 (m, 4H), 4.16 (m, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.82-3.71 (m, 2H), 3.66 (m, 3H), 3.40-3.28 (m, 1H), 3.07 (m, 1H), 2.70-2.57 (m, 1H), 2.47-2.36 (m, 2H), 2.15 (m, 1H), 1.51-1.40 (m, 3H), 1.03-0.87 (m, 11H), 0.77-0.71 (m, 2H), 0.60-0.54 (m, 2H), 0.00 (t, J=3.0Hz, 18H).
(e) ((2S) -1- (((2S) -1- ((4- (8- (3- ((2- cyclopropyl -7- methoxyl group -5- oxo -5,11a- dihydro - 1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -5- oxo -5,11a- Dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- oxopropan -2- base) ammonia Base)-3- methyl-1-oxo-butanes-2- base) carbamic acid (9H- fluorenes-9- base) methyl ester (23)
It under an argon atmosphere, will at -78 DEG C(0.5mL, the 1M in THF) is added dropwise to SEM Double lactams 22 (265mg g, 0.19mmol) are in the solution in THF (10mL).Complete addition in 5 minutes to keep reacting The internal temperature of mixture is constant.After twenty minutes, equal part reaction is quenched with water to analyze for LC/MS, it should be analysis shows that having reacted At.Addition water (20mL) into reaction mixture and removes cryostat.Organic layer is extracted with EtOAc (3x30mL), and uses salt water (50mL) washs combined organic matter, uses MgSO4It dries, filters and solvent is removed by rotary evaporation under reduced pressure.It will slightly produce Object is dissolved in MeOH (12mL), CH2Cl2To form dense stirred suspension in (6mL) and water (2mL) and enough silica gel. After 5 days, suspension is filtered by sinter funnel, and use CH2Cl2/ MeOH (9:1) (200mL) washing is washed completely until product It is de-.Organic layer is washed with salt water (2x70mL), uses MgSO4It dries, filters and solvent is removed by rotary evaporation under reduced pressure.It is logical Cross silica gel column chromatography (100%CHCl3To 96%CHCl3/ 4%MeOH) purifying provide product 23, be yellow solid (162mg, 78%).LC/MS 3.02min (ES+) m/z (relative intensity) 1052.37.
(f) (2S) -2- amino-N- ((2S) -1- ((4- (8- (3- ((2- cyclopropyl -7- methoxyl group -5- oxo -5,11a- Dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -7- methoxyl group -5- oxo - 5,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- oxopropan -2- Base) -3- methylbutyryl amine (17)
Excessive addition piperidines (0.2mL, 2mmol) is to the bis- lactams 23 (76mg, 0.073mmol) of SEM- in DMF (1mL) Solution in.Allow to be stirred at room temperature mixture 20 minutes, at this point, reaction is carried out to completion (monitoring by LC/MS).With CH2Cl2(75mL) releases dilute reaction mixture, and uses H2O (3x75mL) washing organic phase is until completely remove piperidines.With MgSO4Dry organic phase filters under reduced pressure and removes excessive solvent to provide crude product 17, directly under by rotary evaporation It is used in one step.LC/MS 2.32min (ES+) m/z (relative intensity) 830.00.
(g) N- ((2S) -1- (((2S) -1- ((4- (8- (3- ((2- cyclopropyl -7- methoxyl group -5- oxo -5,11a- two Hydrogen -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) oxo-5-7- methoxyl group-5-, 11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza2- yl) phenyl) amino) -1- oxopropan -2- Base) amino)-3- methyl-1-oxo-butanes-2- base)-1- (3- (2,5- dioxo-2,5- dihydro-1H- pyrroles-1- base) third ammonia Base) eight oxa- heptacosane -27- amide (18) of -3,6,9,12,15,18,21,24-.
Under an argon atmosphere, EDCI hydrochloride (14mg, 0.0732mmol) is added to maleimide-PEG8Acid (43.4mg, 0.0732mmol) is in dry CH2Cl2In suspension in (5mL).Addition PBD 17 (60.7mg, Before 0.0732mmol), mixture is stirred at room temperature 1 hour.Lasting stirring completes (usually 5 hours) until reaction.With CH2Cl2Diluting reaction, and with MgSO4H is used before dry2O and salt water washing organic phase filter and pass through rotation under reduced pressure Evaporation removes excessive solvent.By careful silica gel chromatograph (from 100%CHCl3Start to 9:1CHCl3/ MeOH is slowly eluted) Purified product carries out reverse-phase chromatography then to remove unreacted maleimide-PEG8Acid.With 17.6% (21.8mg's) Yield separation product 18.LC/MS 2.57min (ES+) m/z (relative intensity) 1405.30；1H NMR (400MHz, CDCl3)δ 7.91 (t, J=3.5Hz, 1H), 7.80 (d, J=4.0Hz, 1H), 7.75 (d, J=8.8Hz, 1H), 7.69 (d, J=8.7Hz, 1H), 7.54-7.50 (m, 2H), 7.45 (s, 1H), 7.39-7.31 (m, 2H), 6.87 (d, J=10.5Hz, 2H), 6.76 (s, 1H), 6.72-6.68 (m, 2H), 4.74-4.62 (m, 1H), 4.45-4.17 (m, 7H), 3.95 (s, 3H), 3.94 (s, 3H), 3.67-3.58 (m, 34H), 3.54 (m, 2H), 3.42 (dd, J=10.2,5.2Hz, 2H), 3.16-3.07 (m, 1H), 2.92 (dd, J=16.1,4.1Hz, 1H), 2.62-2.49 (m, 4H), 2.48-2.39 (m, 2H), 2.37-2.25 (m, 1H), 1.92 (s, 1H), 1.52-1.44 (m, 3H), 1.10-0.93 (m, 6H), 0.79 (dd, J=9.2,5.3Hz, 2H), 0.57 (dd, J=9.2, 5.3Hz, 2H), NH is not observed.
Embodiment 3
(a) trifluoromethanesulfonic acid (S) -7- methoxyl group -8- (3- (((S) -7- methoxyl group -2- (4- (4- methylpiperazine-1-yl) Phenyl) -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro -1H- pyrrole Cough up simultaneously [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) propoxyl group) -5,11- dioxo -10- ((2- (trimethyl first silicon Alkyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro-1 H-pyrrolo simultaneously [2,1-a] [1,4] benzodiazepine2- base ester (24)((S)-7-methoxy-8-(3-(((S)-7-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)- 5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H- pyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)propoxy)-5,11-dioxo-10-((2- (trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4] benzodiazepin-2-yl trifluoromethanesulfonate)
By Pd (PPh3)4(20.6mg, 0.018mmol) be added to double-enol triflate 12 (500mg, 0.44mmol), N methyl piperazine borate (100mg, 0.4mmol), Na2CO3(218mg, 2.05mmol), MeOH (2.5mL), In the stirring mixture of toluene (5mL) and water (2.5mL).It is small that permission is stirred to react mixture 24 at 30 DEG C in a nitrogen atmosphere When, after the time, all borates are depleted.Then before taking out residual in EtOAc (100mL), evaporation reaction mixing Object uses H to drying2O (2 × 50mL), salt water (50mL) washing, dry (MgSO4), filter and evaporated under reduced pressure to mention For crude product.Pass through purified by flash chromatography (gradient elution: 80:20v/v hexane/EtOAc to 60:40v/v hexane/EtOAc) Product 24 is provided, is weak yellow foam (122.6mg, 25%).
LC/MS 3.15min (ES+) m/z (relative intensity) 1144 ([M+H]+, 20%).
(b) ((S) -1- (((S) -1- ((4- ((S) -7- methoxyl group -8- (3- (((S) -7- methoxyl group -2- (4- (4- methyl Piperazine -1- base) phenyl) -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- Tetrahydro-1 H-pyrrolo simultaneously [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) propoxyl group) -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro-1 H-pyrrolo simultaneously [2,1-a] [1,4] benzodiazepine2- yl) phenyl) amino) -1- oxopropan -2- base) amino) -3- methyl-oxo butane -2- base) carbamic acid (9H- fluorenes - 9- yl) methyl ester (25)
By PBD- triflate 24 (359mg, 0.314mmol), pinacol borate 20 (250mg, 0.408mmol) Toluene/MeOH/H is dissolved in triethylamine (0.35mL, 2.51mmol)2In the mixture of O (2:1:1,3mL).In addition four (three Phenylphosphine) before palladium (0) (21.7mg, 0.018mmol), with argon cleaning and microwave container is filled, and by reaction mixture It is placed in 80 DEG C of microwave 10 minutes.Then, CH is added2Cl2(100mL), and with MgSO4Water is used before dry (2x50mL) and salt water (50mL) wash organic matter, filter and remove volatile matter by rotary evaporation under reduced pressure.Pass through silica gel Chromatographic column (CHCl3/ MeOH, 100% to 9:1) purification of crude product, to provide 25 pure (200mg, 43% yields).LC/MS 3.27min (ES+) m/z (relative intensity) 1478 ([M+H]+, 100%).
(c) ((S) -1- (((S) -1- ((4- ((S) -7- methoxyl group -8- (3- (((S) -7- methoxyl group -2- (4- (4- methyl Piperazine -1- base) phenyl) -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygen Base) propoxyl group) -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) phenyl) ammonia Base)-1- oxopropan-2- base) amino)-3- methyl-1-oxo-butanes-2- base) carbamic acid (9H- fluorenes-9- base) methyl ester (26)
It under an argon atmosphere, will at -78 DEG CThe solution of (0.34mL, the 1M in THF) adds dropwise The bis- lactams 25 (200mg g, 0.135mmol) of SEM- are added in the solution in THF (5mL).In 5 minutes complete addition with Keep the internal temperature of reaction mixture constant.After twenty minutes, equal part reaction is quenched with water to analyze for LC/MS, the analysis is aobvious Show that reaction is completed.Addition water (20mL) into reaction mixture and removes cryostat.With EtOAc (3x30mL) extract organic layer and The organic matter merged is washed with salt water (50mL), uses MgSO4It dries, filters and solvent is removed by rotary evaporation under reduced pressure. Crude product is dissolved in MeOH (6mL), CH2Cl2It is outstanding to form dense stirring in (3mL) and water (1mL) and enough silica gel Supernatant liquid.After 5 days, suspension is filtered by sinter funnel, and use CH2Cl2/ MeOH (9:1) (100mL) is washed until product quilt Elution completely.Organic layer is washed with salt water (2x50mL), uses MgSO4It dries, filters and is removed under reduced pressure by rotary evaporation Solvent.Pass through silica gel column chromatography (100%CHCl3To 96%CHCl3/ 4%MeOH) purifying provides product 26, and it is yellow solid (100mg, 63%).LC/MS 2.67min (ES+) m/z (relative intensity) 1186 ([M+H]+, 5%).
(d) (S) -2- amino-N- ((S) -1- ((4- ((R) -7- methoxyl group -8- (3- (((R) -7- methoxyl group -2- (4- (4- Methylpiperazine-1-yl) phenyl) -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) Oxygroup) propoxyl group) -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) phenyl) ammonia Base) -1- oxopropan -2- base) -3- methylbutyryl amine (27)
Excessive piperidines (0.1mL, 1mmol) is added to PBD 26 (36.4mg, 0.03mmol) in DMF (0.9mL) In solution.Allow to be stirred at room temperature mixture 20 minutes, at this point, reaction is carried out to completion (monitoring by LC/MS).With CH2Cl2(50mL) releases dilute reaction mixture, and uses H2O (3x50mL) washing organic phase is until completely remove piperidines.With MgSO4Dry organic phase filters under reduced pressure and removes excessive solvent to provide crude product 27, directly under by rotary evaporation It is used in one step.LC/MS 2.20min (ES+) m/z (relative intensity) 964 ([M+H]+, 5%).
(e) 6- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base)-N- ((S) -1- (((S) -1- ((4- ((S) -7- Methoxyl group -8- (3- (((S) -7- methoxyl group -2- (4- (4- methylpiperazine-1-yl) phenyl) -5- oxo -5,11a- dihydro -1H- Pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) propoxyl group) -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) phenyl) amino)-1- oxopropan-2- base) amino)-3- methyl-1-oxo Butane -2- base) caproamide (28)
Under an argon atmosphere, EDCI hydrochloride (4.7mg, 0.03mmol) is added to 6- maleimidohexanoic acid (6.5mg, 0.03mmol) is in dry CH2Cl2In suspension in (3mL).Before addition PBD 27 (34mg, thick), in room temperature Lower stirring mixture 1 hour.Lasting stirring completes (6 hours) until reaction.Use CH2Cl2Diluting reaction, and with MgSO4It is dry H is used before2O and salt water washing organic phase filter and remove excessive solvent by rotary evaporation under reduced pressure.By careful Silica gel chromatograph is (from 100%CHCl3Start to 9:1CHCl3/ MeOH is slowly eluted) purified product, carry out reverse-phase chromatography then to remove Remove unreacted maleimide-PEG8Acid.Product 28 has been separated with 41% (14.6mg) yield on two steps.LC/MS 2.40min (ES+) m/z (relative intensity) 1157 ([M+H]+, 5%).
The alternative synthesis of embodiment 4- compound 25
By PBD- triflate 21 (469mg, 0.323mmol), pinacol borate (146.5mg, 0.484mmol) And Na2CO3(157mg, 1.48mmol) is dissolved in toluene/MeOH/H2In the mixture of O (2:1:1,10mL).In addition four (three Phenylphosphine) before palladium (0) (7.41mg, 0.0064mmol), reaction flask is purged with argon gas, and reaction mixture is heated to 30 DEG C overnight.Solvent is removed under reduced pressure, and residual is extracted in H2It is extracted in O (50mL) and with EtOAc (3x50mL). The organic matter merged is washed with salt water (100mL), uses MgSO4It dries, filters and is removed under reduced pressure by rotary evaporation and volatilized Object.Pass through silica gel column chromatography (CHCl3100% to CHCl3/ MeOH 95%:5%) purification of crude product, it is provided with 33% yield 25 pure (885mg).LC/MS 3.27min (ES+) m/z (relative intensity) 1478 ([M+H]+, 100%).
Embodiment 5
(a) (S) -2- (4- aminophenyl) -8- (3- (((S) -2- (benzo [d] [1,3] dioxole -5- base) -7- Methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro -1H- pyrrole Cough up simultaneously [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) propoxyl group) -7- methoxyl group -10- ((2- (trimethyl silyl Base) ethyoxyl) methyl) -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine5,11 (10H, 11aH)-diketone (29)
In a nitrogen atmosphere, by 3,4- (methylene-dioxy) phenylboric acid (356mg, 2.1mmol, 1.3 equivalent), TEA (1.8mL, 12.9mmol, 8 equivalent) and triflate/aniline 13 (1.75g, 1.7mmol, 1 equivalent) are dissolved in ethyl alcohol In the mixture of (7mL), toluene (13mL) and water (2mL).It empties reaction mixture and is adding four (triphenyl phasphine) palladiums (0) (114mg, 0.1mmol, 0.06 equivalent) in the past, is rinsed 3 times with Ar.Flask is emptied again and is rinsed 3 times with Ar, and at 80 DEG C It is heated 8 minutes in microwave, wherein being within 30 seconds pre- mixing time.TLC (80:20v/v ethyl acetate/hexane) analysis instruction starting Material is totally consumed.It is washed with methylene chloride (50mL) diluted reaction mixture and with water (50mL).Use MgSO4Drying is organic Layer, vacuum filter remove solvent.By silica gel column chromatography (60:40 to 20:80v/v hexane/ethyl acetate) purifying provide for The product 29 (1.21g, 71%) of yellow solid.LC/MS(3.92min(ES+) m/z (relative intensity) 1032.44 ([M+H]+, 100)。
(b) (S) -2- (4- aminopropyl) -8- (3- (((S) -2- (benzo [d] [1,3] dioxole -5- base) -7- Methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) propoxyl group)- 7- methoxyl group -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine5 (11aH) -one (30)
Under an ar atmosphere, the bis- lactams 29 (0.25g, 0.24mmol, 1 equivalent) of SEM are dissolved in THF (8mL) and cold But to -78 DEG C.It is added dropwise in 5 minutes(1M in 0.6mL, THF, 2.5 equivalents), while monitoring temperature Degree.After twenty minutes, a small amount of sample is extracted, and checks (work-up) for lcms analysis.It adds water (50mL), removes cryostat, and And solution is washed with ethyl acetate (50mL).Extraction organic layer is simultaneously washed with salt water (60mL), uses MgSO4It is dry, vacuum filter And remove solvent.Crude product is dissolved in EtOH (15mL), CH2Cl2In (7.5mL) and water (2.5mL), and add enough Silica gel is to form dense suspension.After stirring 5 days, filtered by sinter funnel, and use CH2Cl2/MeOH(9:1) (100mL) washing is eluted completely until product.Organic layer is washed with salt water (2x50mL), uses MgSO4Dry, vacuum filter is removed Remove solvent.Pass through silica gel column chromatography (CHCl3, 1% to 4%MeOH gradient) and purify the product 30 provided as yellow solid (94mg, 53%).LC/MS(2.53min(ES+) m/z (relative intensity) 739.64 ([M]+, 70).
(c) ((S) -1- (((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3] dioxole -5- Base) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) third Oxygroup) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) phenyl) Amino)-1- oxopropan-2- base) amino)-3- methyl-1-oxo-butanes-2- base) allyl carbamate (31)
Under an ar atmosphere, stirring alanine-valine-Alloc (180mg, 0.66mmol, 1.2 equivalent) and anhydrous CH2Cl2 EEDQ (163mg, 0.66mmol, 1.2 equivalent) in (21mL) and methanol (1mL).By PBD 30, (407mg, 0.55mmol, 1 work as Amount) it is dissolved in anhydrous CH2Cl2In (21mL) and methanol (1mL), and add it in reaction.After stirring 5 days at room temperature, LC/ MS shows principal product and is formed.Passing through column chromatography (CH2Cl2With 1% to 6%MeOH gradient) purifying before, solvent is removed in vacuum, To be produced as the product 31 (184mg, 34%) of yellow solid.LC/MS(2.95min(ES+) 994.95 ([M of m/z (relative intensity) +H]+, 60).
(d) (S) -2- amino-N- ((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3] dioxane penta Alkene -5- base) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygen Base) propoxyl group) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) Phenyl) amino) -1- oxopropan -2- base) -3- methylbutyryl amine (32)
Under an ar atmosphere, imines 31 (100mg, 0.1mmol, 1 equivalent) is dissolved in anhydrous DCM (10mL) (auxiliary with One drips methanol to help to dissolve).Before being flushed three times in emptying flask and with Ar, be added dropwise pyrrolidines (30 μ L, 0.15mmol, 1.5 equivalents).Before emptying flask and being flushed three times with Ar, Pd (PPh is added3)4(7mg, 6 μm of ol, 0.06 works as Amount).After 1 hour, LC/MS analysis instruction product formation is completely disappeared with starting material.Add Et2O (60mL) is mixed to reaction It closes in object, and stirs it until all products have been precipitated from solution.It is filtered and is precipitated by sinter funnel, and use Et2O washing (2x20mL) twice.Displacement collect flask, and dissolution and by using CHCl3(100mL) sinter washs isolated solid.Very Sky removes solvent to be provided as the crude product 32 of yellow solid, directly uses the crude product 32 in the next step.LC/MS (1.14min(ES+) m/z (relative intensity) 910.40 ([M+H]+, 67).
(e) N- ((S) -1- (((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3] dioxole - 5- yl) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine -8- base) oxygroup) third Oxygroup) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine2- yl) phenyl) Amino)-1- oxopropan-2- base) amino)-3- methyl-1-oxo-butanes-2- base)-1- (3- (bis- oxygroup-2,5- two of 2,5- Hydrogen -1H- pyrroles -1- base) the third amino) eight oxa- heptacosane -27- amide (33) of -3,6,9,12,15,18,21,24-.
Imines 32 (92mg, 0.1mmol, 1.1 equivalent) is dissolved in CHCl3In (6mL), helped by an anhydrous MeOH of drop Hydrotropy solution.Add maleimide-PEG8Sour (53mg, 0.09mmol, 1 equivalent), then add EEDQ (33mg, 0.14mmol, 1.5 equivalents).It is vigorously mixed at room temperature for it at Ar, is formed until LC/MS analysis shows principal product.Vacuum is removed Solvent is removed, and by silica gel column chromatography (CHCl3 and 1% to 10%MeOH gradient) partial purification crude product, generates 33 (81mg). Further by preparative HPLC purified material, to be produced as 33 (26.3mg, 18%) of yellow solid.Quick formic acid operation: LC/MS (1.39min (ES+) m/z (relative intensity) 1485.00 ([M+H]+, 64).
Embodiment 6
(a) ((S) -1- (((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3] dioxo cyclopentene -5- Base) -7- methoxyl group -5,11- dioxo -10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- four Hydrogen -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) propoxyl group) -7- methoxyl group -5,11- dioxo - 10- ((2- (trimethyl silyl) ethyoxyl) methyl) -5,10,11,11a- tetrahydro-1 H-pyrrolo simultaneously [2,1-a] [1,4] benzene And diaza2- yl) phenyl) amino) -1- oxopropan -2- base) amino) -3- methyl-oxo butane -2- base) amino first Sour (9H- fluorenes -9- base) methyl ester (34)
Under an ar atmosphere, by triflate 21 (0.5g, 0.35mmol, 1 equivalent), 3,4- (methylenedioxy) phenyl Boric acid (75mg, 0.45mmol, 1.3 equivalent) and Na2CO3(0.17g, 1.6mmol, 4.5 equivalent) be dissolved in toluene (11mL), In EtOH (5.5mL) and water (5.5mL).Flask is emptied, and is flushed three times with Ar.Add Pd (PPh3)4(24mg, 0.02mmol, 0.06 equivalent), and empty flask again and flushed three times with Ar.It is heated to 30 DEG C and is stirred overnight.LC/ MS analysis shows starting material and completely disappears.Solvent is removed in vacuum, and will residual before being washed with ethyl acetate (60mLx3) It is dissolved in water (60mL).The organic layer combined is washed with salt water (50mL), uses MgSO4Dry, vacuum filter removes solvent.It is logical Cross column chromatography (50:50 to 25:75v/v hexane/ethyl acetate) purifying provide for yellow solid product 34 (310mg, 64%).LC/MS(1.44min(ES-) m/z (relative intensity) 1423.35 ([M-H]-., 79).
(b) ((S) -1- (((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3] dioxole -5- Base) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygroup) third Oxygroup) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) phenyl) Amino)-1- oxopropan-2- base) amino)-3- methyl-1-oxo-butanes-2- base) carbamic acid (9H- fluorenes-9- base) methyl ester (35)
Under an ar atmosphere, the bis- lactams 34 (0.31g, 0.22mmol, 1 equivalent) of SEM are dissolved in THF (10mL) and cold But to -78 DEG C.It is added dropwise in 5 minutes(1M in 0.5mL, THF, 2.5 equivalents) monitor temperature simultaneously Degree.After 30 minutes, a small amount of sample is extracted, and check and analyze for LC/MS.It adds water (50mL), removes cryostat, and with acetic acid second Ester (50mL) washs solution.Extraction organic layer is simultaneously washed with salt water (60mL), uses MgSO4Dry, vacuum filter removes solvent.It will Crude product is dissolved in EtOH (13.2mL), CH2Cl2In (6.6mL) and water (2.2mL), and it is thick to be formed to add enough silica gel Close suspension.After stirring 5 days, filtered by sinter funnel, and use CH2Cl2/ MeOH (9:1) (100mL) washing is until producing Object is eluted completely.Organic layer is washed with salt water (2x50mL), uses MgSO4Dry, vacuum filter removes solvent.Pass through silicagel column Chromatography (CHCl3, 1% to 4%MeOH gradient) purifying provide pure products 35, be yellow solid (185mg, 75%).LC/MS (1.70min(ES+) m/z (relative intensity) 1132.85 ([M+H]+, 60).
(c) (S) -2- amino-N- ((S) -1- ((4- ((S) -8- (3- (((S) -2- (benzo [d] [1,3] dioxane penta Alkene -5- base) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-c] [1,4] benzodiazepine8- yl) oxygen Base) propoxyl group) -7- methoxyl group -5- oxo -5,11a- dihydro -1H- pyrrolo- [2,1-a] [1,4] benzodiazepine2- yl) Phenyl) amino) -1- oxopropan -2- base) -3- methylbutyryl amine (32)
It is before being slowly added piperidines (0.2mL, 2mmol, excessive), imines 35 (82mg, 0.07mmol, 1 equivalent) is molten Solution is in DMF (1mL).The solution is stirred at room temperature 20 minutes, is totally consumed until LC/MS analysis shows starting material. Use CH2Cl2(50mL) diluted reaction mixture is washed with water (50mLx4), uses MgSO4Dry, vacuum filter removes solvent.? Product 33 is used in the case where being further purified without next step.LC/MS(1.15min(ES+) m/z (relative intensity) 910.60([M+H]+, 58).
Embodiment 7
(i) (S)-(2- amino -5- methoxyl group -4- ((triisopropylsilyl) oxygroup) phenyl) (2- (((tert-butyl two Methyl silicane base) oxygroup) methyl) -4- methyl -2,3- dihydro -1H- pyrroles -1- base) ketone (49)
(a) 5- methoxyl group -2- nitro -4- ((triisopropylsilyl) oxygroup) benzaldehyde (42)
Pure tri isopropyl chlorosilane (triisopropylsilylchloride) (56.4mL, 262mmol) is added to miaow In the mixture of azoles (48.7g, 715.23mmol) and 4- hydroxy-5-methyl oxygroup -2- nitrobenzaldehyde 41 (47g, 238mmol) (grinding together).Heating mixture is until phenol and imidazoles melt and become solution (100 DEG C).Allow to be stirred to react mixture 15 Minute, it is then allowed to cool, at that time, observes that solid forms (imidazolitm chloride) in bottom of bottle portion.It is anti-with the dilution of 5%EtOAc/ hexane Answer mixture, and be directly loaded on silica gel, and with 5%EtOAc/ hexane, then with 10%EtOAc/ hexane elution (by In slight excess, very small amount of unreacted TIPSCl is found in the product).With the solution of 5% ethyl acetate in hexane Elute required product.Excessive eluent is removed by rotary evaporation under reduced pressure, is then dried under a high vacuum to provide crystallization Light sensation solid (74.4g, 88%).Purity satisfaction, LC/MS (4.22min (ES+) m/z (relative intensity) 353.88 ([M+H]+., 100))；1H NMR (400MHz, CDCl3) δ 10.43 (s, 1H), 7.60 (s, 1H), 7.40 (s, 1H), 3.96 (s, 3H), 1.35- 1.24 (m, 3H), 1.10 (m, 18H).
(b) 5- methoxyl group -2- nitro -4- ((triisopropylsilyl) oxygroup) benzoic acid (43)
At room temperature, by sodium chlorite (47.3g, 523mmol, 80% industrial grade) and sodium dihydrogen phosphate list alkali (35.2g, 293mmol) (NaH2PO4) solution in water (800mL) is added to compound 2 (74g, 209mmol) in tetrahydro furan In the solution muttered in (500mL).Hydrogen peroxide (60%w/w, 140mL, 2.93mol) is added to the two-phase being vigorously stirred immediately In mixture.Reaction mixture releases gas (oxygen), and the temperature of starting material dissolution and reaction mixture is increased to 45 DEG C. After 30 minutes, LC/MS display reaction is completed.The cooling reaction mixture in ice bath, and hydrochloric acid (1M) is added so that pH is reduced to 3 (that in many cases, it is found that the step is unnecessary, because it has been acid for reacting last pH；It please examine before extraction Look into pH).Then, reaction mixture is extracted with ethyl acetate (1L), is washed with brine organic phase (2 × 100mL) and uses magnesium sulfate It is dry.Organic phase is filtered, and removing excessive solvent by rotary evaporation under reduced pressure to provide quantitative yield is yellow solid Product 43.LC/MS (3.93min (ES-) m/z (relative intensity) 367.74 ([M-H]-., 100))；1H NMR (400MHz, CDCl3) δ 7.36 (s, 1H), 7.24 (s, 1H), 3.93 (s, 3H), 1.34-1.22 (m, 3H), 1.10 (m, 18H).
(c) ((2S, 4R) -2- (((t-butyldimethylsilyl) oxygroup) methyl) -4- hydroxyl pyrrolidine -1- base) (5- methoxyl group -2- nitro -4- ((triisopropylsilyl) oxygroup) phenyl) ketone (45)
DCC (29.2g, 141mmol, 1.2 equivalent) is added to sour 3 (43.5g, 117.8mmol, 1 equivalents) and hydroxyl at 0 DEG C Base benzotriazole hydrate (19.8g, 129.6mmol, 1.1 equivalent) is in the solution in methylene chloride (200mL).Remove cryostat And allow to react and carry out 30 minutes at room temperature, at this point, quickly adding (2S, 4R) -2-t- butyl diformazan under argon gas at -10 DEG C Base silanyloxymethyl -4- hydroxyl pyrrolidine 44 (30g, 129.6mmol, 1.1 equivalent) and triethylamine (24.66mL, 176mmol, 1.5 equivalents) in methylene chloride (100mL) solution (it is a large amount of, can be with by the cooling reaction mixture longer time Shorten the addition time).Allow to be stirred at room temperature reaction mixture 40 minutes to 1 hour, and passes through LC/MS and TLC (EtOAc) Monitoring.Organic phase is washed by the solid that is filtered to remove over celite, and with cold 0.1M HCL aqueous solution, until what is measured PH is 4 or 5.Then organic phase is washed with water, then washs organic phase with saturated sodium bicarbonate aqueous solution.Have with magnesium sulfate drying Machine layer filters and removes excessive solvent by rotary evaporation under reduced pressure.Residue is set to carry out flash column chromatography (silica gel；Gradient: 40/60 ethyl acetate/hexane to 80/20 ethyl acetate/hexane).Excessive solvent is removed by rotary evaporation under reduced pressure to provide Pure products 45 (the slight impure product of the pure products 66% and 17g of 45.5g, in total 90%).LC/MS, 4.23min (ES+) m/ Z (relative intensity) 582.92 ([M+H]+, 100)；1H NMR (400MHz, CDCl3) δ 7.66 (s, 1H), 6.74 (s, 1H), 4.54 (s, 1H), 4.40 (s, 1H), 4.13 (s, 1H), 3.86 (s, 3H), 3.77 (d, J=9.2Hz, 1H), 3.36 (dd, J=11.3, 4.5Hz, 1H), 3.14-3.02 (m, 1H), 2.38-2.28 (m, 1H), 2.10 (ddd, J=13.3,8.4,2.2Hz, 1H), 1.36-1.19 (m, 3H), 1.15-1.05 (m, 18H), 0.91 (s, 9H), 0.17-0.05 (m, 6H), (there are rotational isomers).
(d) (S) -5- (((t-butyldimethylsilyl) oxygroup) methyl) -1- (5- methoxyl group -2- nitro -4- ((three Isopropyl silyl) oxygroup) benzoyl) pyrrolidines -3- ketone (46)
TCCA (8.82g, 40mmol, 0.7 equivalent) is added to 45 (31.7g, 54mmol, 1 equivalents) and TEMPO at 0 DEG C (0.85g, 5.4mmol, 0.1 equivalent) is in the agitating solution in dry methylene chloride (250mL).It is vigorously stirred reaction mixing Object 20 minutes, at this point, TLC (50/50 ethyl acetate/hexane) shows that starting material is totally consumed.It is anti-by diatomite filtering Mixture is answered, and is washed with saturated sodium bicarbonate aqueous solution (100mL), sodium thiosulfate (9g in 300mL), salt water (100mL) Filtrate is washed, and dry with magnesium sulfate.Rotary evaporation provides the product 46 of quantitative yield under reduced pressure.LC/MS 4.52min(ES +) m/z (relative intensity) 581.08 ([M+H]+, 100)；1H NMR (400MHz, CDCl3) δ 7.78-7.60 (m, 1H), 6.85- 6.62 (m, 1H), 4.94 (dd, J=30.8,7.8Hz, 1H), 4.50-4.16 (m, 1H), 3.99-3.82 (m, 3H), 3.80- 3.34 (m, 3H), 2.92-2.17 (m, 2H), 1.40-1.18 (m, 3H), 1.11 (t, J=6.2Hz, 18H), 0.97-0.75 (m, 9H), 0.15-0.06 (m, 6H), (there are rotational isomers).
(e) (S)-trifluoromethanesulfonic acid 5- (((t-butyldimethylsilyl) oxygroup) methyl) -1- (5- methoxyl group -2- Nitro -4- ((triisopropylsilyl) oxygroup) benzoyl) -4,5- dihydro -1H- pyrroles -3- base ester (47)
Under -50 DEG C (acetone/the dry ice bath), there are 2,6- lutidines, (25.6mL, 23.5g, 220mmol, 4 work as It is amount, dry with molecular sieve) in the case where trifluoromethanesulfanhydride anhydride (27.7mL, 46.4g, 165mmol, 3 equivalent) is injected to acutely The ketone 46 (31.9g, 55mmol, 1 equivalent) of stirring is in the suspension in anhydrous methylene chloride (900mL).Allow to be stirred to react Mixture 1.5 hours, fully reacting was shown when LC/MS is at miniature inspection (mini work-up) (water/methylene chloride).By water It is added in still cold reaction mixture, separates organic layer and is washed with saturated sodium bicarbonate, salt water and magnesium sulfate.It filters organic Phase, and excessive solvent is removed by rotary evaporation under reduced pressure.Residual is set to be subjected to flash column chromatography (silica gel；10/90v/v acetic acid Ethyl ester/hexane), it removes excessive eluent and provides product 47 (37.6g, 96%) LC/MS, method 2,4.32min (ES+) m/z (relative intensity) 712.89 ([M+H]+., 100)；1H NMR (400MHz, CDCl3) δ 7.71 (s, 1H), 6.75 (s, 1H), 6.05 (d, J=1.8Hz, 1H), 4.78 (dd, J=9.8,5.5Hz, 1H), 4.15-3.75 (m, 5H), 3.17 (ddd, J=16.2, 10.4,2.3Hz, 1H), 2.99 (ddd, J=16.3,4.0,1.6Hz, 1H), 1.45-1.19 (m, 3H), 1.15-1.08 (m, 18H), 1.05 (s, 6H), 0.95-0.87 (m, 9H), 0.15-0.08 (m, 6H).
(f) (S)-(2- (((t-butyldimethylsilyl) oxygroup) methyl) -4- methyl -2,3- dihydro -1H- pyrroles - 1- yl) (5- methoxyl group -2- nitro -4- ((triisopropylsilyl) oxygroup) phenyl) ketone (48)
Under an argon atmosphere, triphenylarsine (1.71g, 5.60mmol, 0.4 equivalent) is added to triflate 47 (10.00g, 14mmol, 1 equivalent), methyl-boric acid (2.94g, 49.1mmol, 3.5 equivalent), (13g, 56mmol, 4 work as silver oxide Amount) and tripotassium phosphate (17.8g, 84mmol, 6 equivalent) in the mixture in anhydrous dioxanes (80mL).It is anti-with argon cleaning It answers 3 times, and two (benzonitrile) palladium chlorides (II) (540mg, 1.40mmol, 0.1 equivalent) is added.Moment be warming up to 110 DEG C it Before, with argon cleaning reaction 3 times or more (before addition flask, drysyn heat block is warming up to 110 DEG C).After ten minutes, will Reaction is cooled to room temperature, and passes through diatomite filter plates.Solvent is removed by rotary evaporation under reduced pressure.Make to obtain residue It is subjected to flash column chromatography (silica gel；10% ethyl acetate/hexane).It collects and merges pure part, and pass through rotation under reduced pressure Evaporation removes excessive eluent to provide product 48 (4.5g, 55%).LC/MS, 4.27min (ES+) m/z (relative intensity) 579.18([M+H]+., 100)；1H NMR (400MHz, CDCl3) δ 7.70 (s, 1H), 6.77 (s, 1H), 5.51 (d, J= 1.7Hz, 1H), 4.77-4.59 (m, 1H), 3.89 (s, 3H), 2.92-2.65 (m, 1H), 2.55 (d, J=14.8Hz, 1H), 1.62 (d, J=1.1Hz, 3H), 1.40-1.18 (m, 3H), 1.11 (s, 9H), 1.10 (s, 9H), 0.90 (s, 9H), 0.11 (d, J =2.3Hz, 6H).
(g) (S)-(2- amino -5- methoxyl group -4- ((triisopropylsilyl) oxygroup) phenyl) (2- (((tert-butyl two Methyl silicane base) oxygroup) methyl) -4- methyl -2,3- dihydro -1H- pyrroles -1- base) ketone (49)
At about 15 DEG C, zinc powder (28g, 430mmol, 37 equivalent) is added to compound 48 (6.7g, 11.58mmol) and is existed 5% formic acid is in the solution in ethyl alcohol v/v (70mL).Using the heat release that ice bath control generates to protect the temperature of reaction mixture It holds at 30 DEG C or less.After 30 minutes, pass through diatomite filter plates reaction mixture.With ethyl acetate dilute filtrate, with water, Saturated sodium bicarbonate aqueous solution and salt water washing organic phase.With the dry organic phase of magnesium sulfate, filters and pass through rotation under reduced pressure Evaporative removal excessive solvent.The residual made is subjected to flash column chromatography (silica gel；10% ethyl acetate in hexane).It collects and closes And pure part, and excessive solvent is removed to provide product 49 (5.1g, 80%) by rotary evaporation under reduced pressure.LC/MS, 4.23min (ES+) m/z (relative intensity) 550.21 ([M+H]+, 100)；1H NMR (400MHz, CDCl3) δ 7.28 (s, 1H), 6.67 (s, 1H), 6.19 (s, 1H), 4.64-4.53 (m, J=4.1Hz, 1H), 4.17 (s, 1H), 3.87 (s, 1H), 3.77- 3.69 (m, 1H), 3.66 (s, 3H), 2.71-2.60 (m, 1H), 2.53-2.43 (m, 1H), 2.04-1.97 (m, J=11.9Hz, 1H), 1.62 (s, 3H), 1.26-1.13 (m, 3H), 1.08-0.99 (m, 18H), 0.82 (s, 9H), 0.03-0.03 (m, J= 6.2Hz, 6H).
(ii) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -8- ((5- iodo amyl) oxygroup) - 7- methoxyl group -2- methyl -5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10 (5H)-carboxylic acid allyl esters
(a) (S)-(2- (2- (((t-butyldimethylsilyl) oxygroup) methyl) -4- methyl -2,3- dihydro -1H- pyrrole Cough up -1- carbonyl) -4- methoxyl group -5- ((triisopropylsilyl) oxygroup) phenyl) allyl carbamate (50)
Under -78 DEG C (acetone/the dry ice bath), there are anhydrous pyridine (0.48mL, 6.00mmol, 2.2 equivalent) the case where Under, allyl chlorocarbonate (0.30mL, 3.00mmol, 1.1 equivalent) is added to amine 49 (1.5g, 2.73mmol) in anhydrous dichloro In solution in methane (20mL).After 30 minutes, the bath is removed, and reaction mixture is heated to room temperature.Use methylene chloride Diluted reaction mixture, and add the copper sulfate solution of saturation.Then the sodium bicarbonate aqueous solution and salt water of sequence saturation Wash organic layer.With the dry organic phase of magnesium sulfate, filters and excessive solvent is removed to provide production by rotary evaporation under reduced pressure Object 50, products therefrom 50 directly use in next reaction.LC/MS, 4.45min (ES+) m/z (relative intensity) 632.91 ([M+ H]+, 100).
(b) (S)-(2- (2- (hydroxymethyl) -4- methyl -2,3- dihydro -1H- pyrroles -1- carbonyl) -4- methoxyl group -5- ((triisopropylsilyl) oxygroup) phenyl) allyl carbamate (51)
Crude product 50 is dissolved in acetic acid/methanol/tetrahydrofuran/water (28:4:4:8mL) 7:1:1:2 mixture, and It is stirred at room temperature.After 3 hours, starting material is observed by LC/MS and is completely disappeared.With ethyl acetate diluted reaction mixture, And successively with water (2 × 500mL), saturated sodium bicarbonate (200mL) and salt water washing.With the dry organic phase of magnesium sulfate, filtering is simultaneously Excess ethyl acetate is removed by rotary evaporation under reduced pressure.The residue made is subjected to flash column chromatography (silica gel；In hexane 25% ethyl acetate).It collects and merges pure part and excessive eluent is removed to provide the phase by rotary evaporation under reduced pressure The product 51 (1g, 71%) of prestige.LC/MS, 3.70min (ES+) m/z (relative intensity) 519.13 ([M+H]+., 95)；1H NMR (400MHz, CDCl3) δ 8.34 (s, 1H), 7.69 (s, 1H), 6.78 (s, 1H), 6.15 (s, 1H), 5.95 (ddt, J=17.2, 10.5,5.7Hz, 1H), 5.33 (dq, J=17.2,1.5Hz, 1H), 5.23 (ddd, J=10.4,2.6,1.3Hz, 1H), 4.73 (tt, J=7.8,4.8Hz, 1H), 4.63 (dt, J=5.7,1.4Hz, 2H), 4.54 (s, 1H), 3.89-3.70 (m, 5H), 2.87 (dd, J=16.5,10.5Hz, 1H), 2.19 (dd, J=16.8,4.6Hz, 1H), 1.70 (d, J=1.3Hz, 3H), 1.38- 1.23 (m, 3H), 1.12 (s, 10H), 1.10 (s, 8H).
(c) (11S, 11aS) -11- hydroxyl -7- methoxyl group -2- methyl -5- oxo -8- ((triisopropylsilyl) oxygen Base) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid allyl esters (52)
Under an argon, under -78 DEG C (dry ice/acetone batch), by dimethyl sulfoxide (0.35mL, 4.83mmol, 2.5 equivalent) It is added dropwise to oxalyl chloride (0.2mL, 2.32mmol, 1.2 equivalent) in the solution in anhydrous methylene chloride (10mL).10 minutes Afterwards, still at -78 DEG C, it is slowly added solution of 51 (1g, the 1.93mmol) in anhydrous methylene chloride (8mL).After 15 minutes, by (1.35mL passes through drop addition triethylamineMolecular sieve is dry, 9.65mmol, 5 equivalents) and remove dry ice/acetone batch.Make to react Mixture reaches room temperature and is extracted with cold hydrochloric acid (0.1M), saturated sodium bicarbonate aqueous solution and salt water.It is organic with magnesium sulfate drying Phase filters and removes excessive methylene chloride by rotary evaporation under reduced pressure to provide product 52 (658mg, 66%).LC/MS, 3.52min (ES+) m/z (relative intensity) (400MHz, CDCl3) δ 7.20 (s, 1H), 6.75-6.63 (m, J=8.8,4.0Hz, 2H), 5.89-5.64 (m, J=9.6,4.1Hz, 2H), 5.23-5.03 (m, 2H), 4.68-4.38 (m, 2H), 3.84 (s, 3H), 3.83-3.77 (m, 1H), 3.40 (s, 1H), 3.05-2.83 (m, 1H), 2.59 (d, J=17.1Hz, 1H), 1.78 (d, J= 1.3Hz, 3H), 1.33-1.16 (m, 3H), 1.09 (d, J=2.2Hz, 9H), 1.07 (d, J=2.1Hz, 9H).
(d) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -7- methoxyl group -2- methyl -5- oxo - 8- ((triisopropylsilyl) oxygroup) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10 (5H)-carboxylic acid allyl esters (53)
Under argon gas, at 0 DEG C, by t-butyldimethylsilyl triflate, (0.70mL, 3.00mmol, 3 work as Amount) compound 52 (520mg, 1.00mmol) and 2 are added to, 6- lutidines (0.46mL, 4.00mmol, 4 equivalent) is in nothing In solution in water methylene chloride (40mL).After 10min, removes cryostat and reaction mixture is stirred at room temperature 1 hour.With Water, saturated sodium bicarbonate aqueous solution and salt water extract reaction mixture.With the dry organic phase of magnesium sulfate, filters and lead under reduced pressure It crosses rotary evaporation and removes excessive solvent.The residual made is subjected to flash column chromatography (silica gel；Gradient, 10% acetic acid second in hexane Ester 20% ethyl acetate into hexane).It collects and merges pure part and excessive elution is removed by rotary evaporation under reduced pressure Liquid is to obtain product 53 (540mg, 85%).LC/MS, 4.42min (ES+) m/z (relative intensity) 653.14 ([M+Na]+., 100)；1H NMR (400MHz, CDCl3) δ 7.20 (s, 1H), 6.71-6.64 (m, J=5.5Hz, 2H), 5.83 (d, J=9.0Hz, 1H), 5.80-5.68 (m, J=5.9Hz, 1H), 5.14-5.06 (m, 2H), 4.58 (dd, J=13.2,5.2Hz, 1H), 4.36 (dd, J=13.3,5.5Hz, 1H), 3.84 (s, 3H), 3.71 (td, J=10.1,3.8Hz, 1H), 2.91 (dd, J=16.9, 10.3Hz, 1H), 2.36 (d, J=16.8Hz, 1H), 1.75 (s, 3H), 1.31-1.16 (m, 3H), 1.12-1.01 (m, J= 7.4,2.1Hz, 18H), 0.89-0.81 (m, 9H), 0.25 (s, 3H), 0.19 (s, 3H).
(e) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -8- hydroxyl -7- methoxyl group -2- methyl - 5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid allyl esters (54)
Lithium acetate (87mg, 0.85mmol) is added to compound 53 (540mg, 0.85mmol) in aqueous dimethyl formyl In solution in amine (6mL, 50:1DMF/ water).After 4 hours, fully reacting, with ethyl acetate (25mL) diluted reaction mixture And with aqueous citric acid solution (about pH 3), water and salt water washing.With the dry organic layer of magnesium sulfate, filters and pass through rotation under reduced pressure Turn evaporation and removes Excess ethyl acetate.The residue made is subjected to flash column chromatography (silica gel；Gradient, in hexane 25% to 75% ethyl acetate).It collects and merges pure part and excessive eluent is removed to be produced by rotary evaporation under reduced pressure Object 54 (400mg, quantitative).LC/MS, (3.33min (ES+) m/z (relative intensity) 475.26 ([M+H]+, 100).
(f) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -8- ((5- iodo amyl) oxygroup) -7- Methoxyl group -2- methyl -5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10(5H)- Carboxylic acid allyl esters (55)
Two iodopentanes (0.63mL, 4.21mmol, 5 equivalents) and potassium carbonate (116mg, 0.84mmol, 1 equivalent) are added to Phenol 54 (400mg, 0.84mmol) is in the solution in acetone (4mL, being dried with molecular sieve).Then, by reaction mixture plus Heat is to 60 DEG C and stirs 6 hours.Acetone is removed by rotary evaporation under reduced pressure.The residue made is subjected to flash column chromatography (silica gel；50/50, v/v, hexane/ethyl acetate).It collects and merges pure part and remove excessive eluent to provide 90% production The 55 of rate.LC/MS, 3.90min (ES+) m/z (relative intensity) 670.91 ([M]+, 100).1H NMR (400MHz, CDCl3)δ 7.23 (s, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 5.87 (d, J=8.8Hz, 1H), 5.83-5.68 (m, J=5.6Hz, 1H), 5.15-5.01 (m, 2H), 4.67-4.58 (m, 1H), 4.45-4.35 (m, 1H), 4.04-3.93 (m, 2H), 3.91 (s, 3H), 3.73 (td, J=10.0,3.8Hz, 1H), 3.25-3.14 (m, J=8.5,7.0Hz, 2H), 2.92 (dd, J=16.8, 10.3Hz, 1H), 2.38 (d, J=16.8Hz, 1H), 1.95-1.81 (m, 4H), 1.77 (s, 3H), 1.64-1.49 (m, 2H), 0.88 (s, 9H), 0.25 (s, 3H), 0.23 (s, 3H).
(iii) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -8- hydroxyl -7- methoxyl group -2- first Base -5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10 (5H)-carboxylic acid 4- (2- (1- ((1- (allyloxy) -4- methyl-1,2- dioxo pentane -3- base) amino) -1- oxopropan -2- base) diazanyl) benzyl Ester (70)
(a) 3- (2- (2- (4- ((((2- ((S) -2- (((t-butyldimethylsilyl) oxygroup) methyl) -4- methyl - 2,3- dihydro -1H- pyrroles -1- carbonyl) -4- methoxyl group -5- ((triisopropylsilyl) oxygroup) phenyl) carbamoyl) Oxygroup) methyl) phenyl) diazanyl) the third amino) -4- methyl -2- oxopentanoic acid allyl ester (56)
At 5 DEG C (ice baths), triethylamine (2.23mL, 18.04mmol, 2.2 equivalent) is added to amine 49 (4g, 8.20mmol) With triphosgene (778mg, 2.95mmol, 0.36 equivalent) in the agitating solution in anhydrous tetrahydro furan (40mL).By from anti- It answers mixture interval to remove part, terminated with methanol and carries out LC/MS analysis to monitor the progress of isocyanates reaction.Once different The formation of cyanate is completed, then by injecting alloc-Val-Ala-PABOH (4.12g, 12.30mmol, 1.5 equivalent) and three Solution of the ethamine (1.52ml, 12.30mmol, 1.5 equivalent) in anhydrous tetrahydro furan (40mL) is quickly added to fresh preparation Isocyanates in.Then mixture is stirred to react 4 hours at 40 DEG C.Excessive solvent is removed by rotary evaporation under reduced pressure. The residue made is subjected to flash column chromatography (silica gel, gradient, 1% methanol to 5% methanol in chloroform).(use EtOAc and oneself The substitution chromatographic condition of alkane is also successful).It collects and merges pure part and removed excessively by rotary evaporation under reduced pressure Eluent is to obtain product 56 (3.9g, 50%).LC/MS, 4.23min (ES+) m/z (relative intensity) 952.36 ([M+H]+., 100)；1H NMR (400MHz, CDCl3) δ 8.62 (br s, 1H), 8.46 (s, 1H), 7.77 (br s, 1H), 7.53 (d, J= 8.4Hz, 2H), 7.32 (d, J=8.5Hz, 2H), 6.76 (s, 1H), 6.57 (d, J=7.6Hz, 1H), 6.17 (s, 1H), 6.03- 5.83 (m, 1H), 5.26 (dd, J=33.8,13.5Hz, 3H), 5.10 (s, 2H), 4.70-4.60 (m, 2H), 4.58 (dd, J= 5.7,1.3Hz, 2H), 4.06-3.99 (m, 1H), 3.92 (s, 1H), 3.82-3.71 (m, 1H), 3.75 (s, 3H), 2.79-2.64 (m, 1H), 2.54 (d, J=12.9Hz, 1H), 2.16 (dq, J=13.5,6.7Hz, 1H), 1.67 (s, 3H), 1.46 (d, J= 7.0Hz, 3H), 1.35-1.24 (m, 3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.97 (d, J=6.8Hz, 3H), 0.94 (d, J =6.8Hz, 3H), 0.87 (s, 9H), 0.07-0.02 (m, 6H).
(b) 3- (2- (2- (4- ((((2- ((S) -2- (hydroxymethyl) -4- methyl -2,3- dihydro -1H- pyrroles's -1- carbonyl Base) -4- methoxyl group -5- ((triisopropylsilyl) oxygroup) phenyl) carbamoyl) oxygroup) methyl) phenyl) diazanyl) third Amino) -4- methyl -2- oxopentanoic acid allyl ester (57)
TBS ether 56 (1.32g, 1.38mmol) is dissolved in acetic acid/methanol/tetrahydrofuran/water (14:2:2:4mL) 7: In 1:1:2 mixture and allow to be stirred at room temperature.After 3 hours, starting material is not observed by LC/MS.Use ethyl acetate (25mL) diluted reaction mixture and sequence water, saturated sodium bicarbonate aqueous solution and salt water washing.It is organic with magnesium sulfate drying Phase filters and removes Excess ethyl acetate by rotary evaporation under reduced pressure.The residue made carries out flash column chromatography (silicon Glue；2% ethanol/methylene).Collect and merge pure part and under reduced pressure by rotary evaporation remove excessive eluent with Product 57 (920mg, 80%) needed for providing.LC/MS, 3.60min (ES+) m/z (relative intensity) 838.18 ([M+H]+., 100)。1H NMR (400MHz, CDCl3) δ 8.55 (s, 1H), 8.35 (s, 1H), 7.68 (s, 1H), 7.52 (d, J=8.1Hz, 2H), 7.31 (d, J=8.4Hz, 2H), 6.77 (s, 1H), 6.71 (d, J=7.5Hz, 1H), 6.13 (s, 1H), 5.97-5.82 (m, J=5.7Hz, 1H), 5.41-5.15 (m, 3H), 5.10 (d, J=3.5Hz, 2H), 4.76-4.42 (m, 5H), 4.03 (t, J =6.6Hz, 1H), 3.77 (s, 5H), 2.84 (dd, J=16.7,10.4Hz, 1H), 2.26-2.08 (m, 2H), 1.68 (s, 3H), 1.44 (d, J=7.0Hz, 3H), 1.30 (dt, J=14.7,7.4Hz, 3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.96 (d, J =6.8Hz, 3H), 0.93 (d, J=6.8Hz, 3H).
(c) (11S, 11aS) -11- hydroxyl -7- methoxyl group -2- methyl -5- oxo -8- ((triisopropylsilyl) oxygen Base) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid 4- (2- (1- ((1- (allyloxy) -4- methyl-1,2- dioxo pentane -3- base) amino) -1- oxopropan -2- base) diazanyl) benzyl ester (58)
Under an argon, -78 DEG C (dry ice/acetone batch) by dimethyl sulfoxide (0.2mL, 2.75mmol, 2.5 equivalent) dropwise Oxalyl chloride (0.11mL, 1.32mmol, 1.2 equivalent) is added in the solution in anhydrous methylene chloride (7mL).After ten minutes, Still at -78 DEG C, it is slowly added solution of 57 (920mg, the 1.10mmol) in anhydrous methylene chloride (5mL).After 15 minutes, by (0.77mL passes through drop addition triethylamineMolecular sieve is dry, 5.50mmol, 5 equivalents) and remove dry ice/acetone batch.Make to react Mixture reaches room temperature and is extracted with cold hydrochloric acid (0.1M), saturated sodium bicarbonate aqueous solution and salt water.Have by magnesium sulfate drying Machine phase filters and removes excessive methylene chloride by rotary evaporation under reduced pressure.The residue made is subjected to quick column Chromatography (silica gel, gradient, 2% methanol to 5% methanol in methylene chloride).It collects and merges pure part and pass through rotation under reduced pressure Turn evaporation and removes excessive eluent to provide product 58 (550mg, 60%).LC/MS, 3.43min (ES+) m/z (relative intensity) 836.01([M]+, 100).1H NMR (400MHz, CDCl3) δ 8.39 (s, 1H), 7.52-7.40 (m, 2H), 7.21-7.08 (m, J =11.5Hz, 2H), 6.67 (s, 1H), 6.60-6.47 (m, J=7.4Hz, 1H), 5.97-5.83 (m, 1H), 5.79-5.66 (m, 1H), 5.38-4.90 (m, 6H), 4.68-4.52 (m, J=18.4,5.5Hz, 4H), 4.04-3.94 (m, J=6.5Hz, 1H), 3.87-3.76 (m, 5H), 3.00-2.88 (m, 1H), 2.66-2.49 (m, 2H), 2.21-2.08 (m, 2H), 1.76 (s, 3H), 1.45 (d, J=7.0Hz, 3H), 1.09-0.98 (m, J=8.9Hz, 18H), 0.96 (d, J=6.7Hz, 3H), 0.93 (d, J= 6.9Hz, 3H).
(d) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -7- methoxyl group -2- methyl -5- oxo - 8- ((triisopropylsilyl) oxygroup) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10 (5H)-carboxylic acid 4- (2- (1- ((1- (allyloxy) -4- methyl-1,2- dioxo pentane -3- base) amino) -1- oxo third Alkane -2- base) diazanyl) benzyl ester (59)
Under argon gas, at 0 DEG C, by t-butyldimethylsilyl triflate, (0.38mL, 1.62mmol, 3 work as Amount) compound 58 (450mg, 0.54mmol) and 2 are added to, 6- lutidines (0.25mL, 2.16mmol, 4 equivalent) is in nothing In solution in water methylene chloride (5mL).After 10min, removes cryostat and reaction mixture is stirred at room temperature 1 hour.With Water, saturated sodium bicarbonate aqueous solution and salt water extract reaction mixture.With the dry organic phase of magnesium sulfate, filters and lead under reduced pressure It crosses rotary evaporation and removes excessive solvent.The residual made is subjected to flash column chromatography (silica gel；50/50v/v hexane/acetic acid second Ester).Collect and merge pure part and excessive eluent removed by rotary evaporation under reduced pressure with obtain product 59 (334mg, 65%).LC/MS, 4.18min (ES+) m/z (relative intensity) 950.50 ([M]+, 100).1H NMR (400MHz, CDCl3)δ 8.53 (s, 1H), 8.02 (s, 1H), 7.44 (d, J=7.6Hz, 2H), 7.21 (s, 1H), 7.08 (d, J=8.2Hz, 2H), 6.72-6.61 (m, J=8.9Hz, 2H), 6.16 (s, 1H), 5.97-5.79 (m, J=24.4,7.5Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5Hz, 1H), 4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7Hz, 1H), 3.87 (s, 3H), 3.74 (td, J=9.6,3.6Hz, 1H), 2.43-2.09 (m, J=34.8,19.4,11.7Hz, 3H), 1.76 (s, 3H), 1.43 (d, J=6.9Hz, 3H), 1.30-1.21 (m, 3H), 0.97 (d, J=6.7Hz, 3H), 0.92 (t, J= 8.4Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H), 0.12 (s, 3H).
(e) (11S, 11aS) -11- ((t-butyldimethylsilyl) oxygroup) -8- hydroxyl -7- methoxyl group -2- methyl - 5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid 4- (2- (1- ((1- (allyloxy) -4- methyl-1,2- dioxo pentane -3- base) amino) -1- oxopropan -2- base) diazanyl) benzyl ester (60)
Lithium acetate (50mg, 0.49mmol) is added to compound 59 (470mg, 0.49mmol) in aqueous dimethyl formyl In solution in amine (4mL, 50:1DMF/ water).After 4 hours, fully reacting with ethyl acetate diluted reaction mixture and uses lemon Lemon acid (about pH 3), water and salt water washing.With the dry organic layer of magnesium sulfate, filters and removed under reduced pressure by rotary evaporation Measure ethyl acetate.The residue made is subjected to flash column chromatography (silica gel；Gradient, 50/50 to 25/75v/v hexane/acetic acid second Ester).Collect and merge pure part and excessive eluent removed by rotary evaporation under reduced pressure with obtain product 60 (400mg, It is quantitative).LC/MS, 3.32min (ES+) m/z (relative intensity) 794.18 ([M+H]+, 100).1H NMR (400MHz, CDCl3)δ 8.53 (s, 1H), 8.02 (s, 1H), 7.44 (d, J=7.6Hz, 2H), 7.21 (s, 1H), 7.08 (d, J=8.2Hz, 2H), 6.72-6.61 (m, J=8.9Hz, 2H), 6.16 (s, 1H), 5.97-5.79 (m, J=24.4,7.5Hz, 2H), 5.41-5.08 (m, 5H), 4.86 (d, J=12.5Hz, 1H), 4.69-4.60 (m, 1H), 4.57 (s, 1H), 4.03 (t, J=6.7Hz, 1H), 3.87 (s, 3H), 3.74 (td, J=9.6,3.6Hz, 1H), 2.43-2.09 (m, J=34.8,19.4,11.7Hz, 3H), 1.76 (s, 3H), 1.43 (d, J=6.9Hz, 3H), 1.30-1.21 (m, 3H), 0.97 (d, J=6.7Hz, 3H), 0.92 (t, J= 8.4Hz, 3H), 0.84 (s, 9H), 0.23 (s, 3H), 0.12 (s, 3H).
(iv) (11S, 11aS) -11- hydroxyl -7- methoxyl group -8- ((5- ((oxo -5 (S) -7- methoxyl group -2- methyl -5-, 11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 8- yl) oxygroup) amyl) oxygroup) -2- methyl -5- Oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza - 10 (5H)-carboxylic acid 4- ((2S, 5S)- 37- (2,5- dioxo-2,5- dihydro-1H- pyrroles-1- base) trioxy--10,13,16-5- isopropyl-2- methyl-4,7,35-, Eight oxa- -3,6,34- of 19,22,25,28,31-, three azepine heptatriacontane amino) benzyl ester (64) ((11S, 11aS) -4- ((2S, 5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35- trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatri acontanamido) benzyl11-hydroxy-7-methoxy-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a- dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl- 5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate)
(a) (11S) -8- ((5- (((11S) -10- (((4- (2- (1- ((1- (allyloxy) -4- methyl-1,2- dioxo Pentane -3- base) amino) -1- oxopropan -2- base) diazanyl) benzyl) oxygroup) carbonyl) -11- ((t-butyl-dimethylsilyl Base) oxygroup) -7- methoxyl group -2- methyl -5- oxo -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] Diaza8- yl) oxygroup) amyl) oxygroup) -11- ((t-butyldimethylsilyl) oxygroup) -7- methoxyl group -2- first Base -5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid allyls Ester (61)
Potassium carbonate (70mg, 0.504mmol, 1 equivalent) is added to 55 (370mg, 0.552mmol, 1.2 equivalents) and phenol 60 (400mg, 0.504mmol) are in the solution in dry acetone (25mL).It is stirred to react at 70 DEG C 8 hours.LC/MS shows institute There is starting material not consume, therefore reaction is made to be stirred at room temperature overnight and be stirred for 2 hours at second day.Pass through under reduced pressure Rotary evaporation removes acetone.The residue made is subjected to flash column chromatography (silica gel；80% ethyl acetate is to 100% in hexane Ethyl acetate).It collects and merges pure part and excessive eluent is removed to obtain product 61 by rotary evaporation under reduced pressure (385mg, 57%).LC/MS, 4.07min (ES+) m/z (relative intensity) 1336.55 ([M+H]+, 50).
(b) (11S) -8- ((5- (((11S) -10- (((4- (2- (1- ((1- (allyloxy) -4- methyl-1,2- dioxo Pentane -3- base) amino) -1- oxopropan -2- base) diazanyl) benzyl) oxygroup) carbonyl) -11- hydroxyl -7- methoxyl group -2- methyl - 5- oxo -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) amyl) Oxygroup) -11- hydroxyl -7- methoxyl group -2- methyl -5- oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] Diaza10 (5H)-carboxylic acid allyl esters (62)
Four n-butylamine fluorides (1M, 0.34mL, 0.34mmol, 2 equivalent) are added to 61 (230mg, 0.172mmol) to exist In solution in anhydrous tetrahydro furan (3mL).After ten minutes, starting material is completely exhausted.It is diluted with ethyl acetate (30mL) Reaction mixture and with water and salt water sequential purge.With the dry organic phase of magnesium sulfate, filters and pass through rotary evaporation under reduced pressure Remove Excess ethyl acetate.Obtained residue 62 is used as to the crude mixture of next reaction.LC/MS, 42.87min (ES+) m/ Z (relative intensity) 1108.11 ([M+H]+, 100).
(c) (11S) -11- hydroxyl -7- methoxyl group -8- ((5- ((7- methoxyl group -2- methyl -5- oxo -5,11a- dihydro - 1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) amyl) oxygroup) oxo-11-2- methyl-5-, 11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid 4- (2- (1- ((1- amino -3- Methyl-1-oxo-butanes-2- base) amino)-1- oxopropan-2- base) diazanyl) benzyl ester (63)
Tetrakis triphenylphosphine palladium (0) (12mg, 0.01mmol, 0.06 equivalent) is added to thick 62 (0.172mmol) and pyrrole Alkane (36 μ L, 0.43mmol, 2.5 equivalent) is coughed up in the solution in anhydrous methylene chloride (10mL).Mixture 20 is stirred to react to divide The dilution of Zhong Bingyong methylene chloride, and sequence saturated aqueous ammonium chloride and salt water washing.Organic phase is dried using magnesium sulfate, It filters and excessive methylene chloride is removed by rotary evaporation under reduced pressure.Obtained residue 63 is used as the thick of next reaction Mixture.LC/MS, 42.38min (ES+) m/z (relative intensity) 922.16 ([M+H]+, 40).
(d) (11S, 11aS) -11- hydroxyl -7- methoxyl group -8- ((5- ((oxo -5 (S) -7- methoxyl group -2- methyl -5-, 11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) amyl) oxygroup) -2- methyl -5- Oxo -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza- 10 (5H)-carboxylic acid 4- ((2S, 5S)- 37- (2,5- dioxo-2,5- dihydro-1H- pyrroles-1- base) trioxy--10,13,16-5- isopropyl-2- methyl-4,7,35-, Eight oxa- -3,6,34- of 19,22,25,28,31-, three azepine heptatriacontane amino) benzyl ester (64)
1- ethyl -3- (3 '-dimethyl aminopropyl) carbodiimide (EDCI, 33mg, 0.172mmol) is added to thick 63 (0.172mmol) and Mal- (PEG)8Sour (100mg, 0.172mmol) is in the solution in anhydrous methylene chloride (10mL).It stirs It mixes reaction 2 hours and cannot observe the presence of starting material again by LC/MS.With methylene chloride diluting reaction and with water and Salt water sequential purge.Using the dry organic phase of magnesium sulfate, filters and excessive dichloromethane is removed by rotary evaporation under reduced pressure Alkane.The residue made is subjected to flash column chromatography (silica gel, 100% chloroform, 10% methanol into chloroform).It collects and merges pure Part and excessive eluent removed by rotary evaporation under reduced pressure (25%) 60mg, 3 step yields is to provide 64 (E).
Embodiment 8
Compound 65 is the compound 79 of WO 2011/130598.
(11S) -11- hydroxyl -7- methoxyl group -8- (3- ((7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkene -1- base) -5, 11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- yl) oxygroup) propoxyl group) -5- oxo -2- ((E) -propyl- 1- alkene -1- base) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylics Sour 4- (four oxa- -3,19,22- of 1- iodo -20- isopropyl -23- methyl -2,18,21- trioxy- -6,9,12,15-, three azepine Lignocerane amino) benzyl ester (66) ((11S) -4- (1-iodo-20-isopropyl-23-methyl-2,18,21- trioxo-6,9,12,15-tetraoxa-3,19,22-triazatetracosanamido)benzyl11-hydroxy-7- methoxy-8-(3-((7-methoxy-5-oxo-2-((E)-prop-1-en-1-yl)-5,11a-dihydro-1H-benzo [e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-5-oxo-2-((E)-prop-1-en-1- yl)-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)- carboxylate)
By N, N '-diisopropylcarbodiimide (DIC, 4.71 μ L, 0.0304mmol) is added to amine 65 (0.0276mmol) With iodo-(PEG)4Sour (13.1mg, 0.0304mmol) is in the solution in anhydrous methylene chloride (0.8mL).It is stirred to react 3 Hour and the presence that cannot observe starting material again by LC/MS.Reaction mixture is directly loaded to thin-layer chromatography (TLC) it is purified on plate and by prep-TLC (10% methanol in chloroform).Pure band is scraped from TLC plate, is extracted in chloroform In 10% methanol, filters and excessive solvent is removed to obtain 66 (D) (20.9mg, 56%) by rotary evaporation under reduced pressure.LC/ MS, method 2,3.08min (ES+) m/z (relative intensity) 1361.16 ([M+H]+, 100).
The General Experimental Procedures of embodiment 9
Using the 1200 series LC/MS of Agilent with 6110 quadrupole MS of Agilent, obtained with electrospray ionization Obtain LCMS data.0.1% acetic acid of mobile phase A-in water.Mobile phase B-in acetonitrile 0.1%.Flow velocity is 1.00ml/ points Clock.Gradient is to rise to 95%B from 5%B through 3 minutes, is maintained at 95%B lower 1 minute, then fell back to 5%B through 6 seconds.Total fortune The row time is 5 minutes.Column: Phenomenex Gemini-NX 3 μm of C18,30 × 2.00mm.Based on the UV inspection at 254nm The tomographic map of survey.Mass spectrum is obtained using MS and with cation mode (positive mode).Made based on δ scale with 400MHz With Bruker AV400, to measure proton nmr chemical shift value.Following abbreviation: s has been used, it is unimodal；D, it is bimodal；T, three Weight peak；Q, quartet；M, multiplet；Br, it is wide.Coupling constant is reported as unit of Hz.Unless otherwise indicated, Merck is used Kieselgel silica (Art.9385) carries out column chromatography (passing through fast procedure).It is separated using coupling in Waters 2795HPLC The Waters Micromass LCT instrument of module acquires mass spectrography (MS) data.With silica gel aluminium sheet (Merck 60, F254) into Row thin-layer chromatography (TLC).Every other chemicals and solvent purchased from Sigma-Aldrich or Fisher Scientific and Further purifying is used without such as supply.
Optical activity (Optical is measured with 220 polarimeter of ADP (Bellingham Stanley Ltd.) Rotation concentration (c) is provided) and as unit of g/100mL.It is measured using numeral melting point instrument (Electrothermal) Fusing point.IR spectrum is recorded with Perkin-Elmer Spectrum 1000FT IR spectrometer.Bruker is used at 300k Avance NMR spectrometer obtains at 400 and 100MHz respectively1H and13C H NMR spectroscopy.It is reported relative to TMS (δ=0.0ppm) Chemical shift is accused, and signal is designated as s (unimodal), d (bimodal), t (triplet), dt (double triplets), dd (bimodal pair Peak), ddd (bimodal double doublet) or m (multiplet), and be that unit provides coupling constant with hertz (Hz).Using coupling in tool There is the Waters MicromassZQ instrument of the Waters 2695HPLC of Waters 2996PDA to acquire mass spectrography (MS) number According to.The Waters Micromass ZQ parameter used is: capillary pressure (kV), 3.38；Cone (V), 35；Extractor (V), 3.0；Source temperature (DEG C), 100；Desolvation temperature (DEG C), 200；Cone flow rate (L/h), 50；Desolvation flow velocity (L/ H), 250.High resolution mass spec method is recorded with Waters Micromass QTOF Global and with cationic W mode (HRMS) data, wherein introducing the sample into instrument using the Pyrex tip of metal coating.With silica gel aluminium sheet (Merck 60,F254) thin-layer chromatography (TLC) is carried out, and with silica gel (Merck 60,230-400 mesh ASTM) Lai Jinhang flash chromatography.It removes Except HOBt (NovaBiochem) and supported reagents (Argonaut), every other chemicals and solvent are purchased from Sigma- Aldrich is simultaneously used without further purifying such as supply.Under a dry nitrogen atmosphere, having existing for appropriate desiccant Under the conditions of, prepare anhydrous solvent by distilling, andIt is stored on molecular sieve or sodium wire.Petroleum ether refers to boil at 40-60 DEG C The fraction risen.
General LC/MS condition: it is run using the mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%) HPLC(Waters Alliance 2695).Gradient: Initial Composition 5%B, through 1.0 minutes, then 5%B was arrived in 3 minutes 95%B.At 95%B, composition is kept 0.5 minute, returned to 5%B in 0.3 minute originally.Total gradient runing time is equal to 5 points Clock.Flow velocity is 3.0mL/ minutes, separates 400 μ L via zero dead volume T shape pipe, is passed into mass spectrograph.Wavelength detection range: 220 to 400nm.Function type: diode array (535 scanning).Column: Onyx Monolithic C1850×4.60mm。
Embodiment 9
(i) main intermediate
(a)
(a-i) (S) -2- (allyloxycarbonyl amino) -3 Methylbutanoic acid (I2)
Allyl chlorocarbonate (36.2mL, 340.59mmol, 1.2 equivalent) is added dropwise to Valine (I1) (33.25g, 283.82mmol, 1.0 equivalent) and potassium carbonate (59.27g, 425.74mmol, 1.5 equivalent) in water (650mL) and In agitating solution in THF (650mL).Reaction mixture is stirred at room temperature 18 hours, solvent is then concentrated under reduced pressure and is used in combination Diethyl ether (3 × 100mL) extracts surplus solution.Aqueous fractions are acidified to pH 2 with dense HCl and are extracted with DCM (3 × 100mL) It takes.It is washed with brine the organic matter of merging, uses MgSO4It dries, filters and is concentrated under reduced pressure to be provided as the product of colorless oil (57.1g, it is assumed that yield 100%).LC/MS(1.966min(ES+)), m/z:202.1 [M+H]+。1H NMR (400MHz, DMSO- d6) δ 12.57 (br s, 1H), 7.43 (d, 1H, J=8.6Hz), 5.96-5.86 (m, 1H), 5.30 (ddd, 1H, J=17.2, 3.4,1.7Hz), 5.18 (ddd, 1H, J=10.4,2.9,1.6Hz), 4.48 (dt, 2H, J=5.3,1.5Hz), 3.85 (dd, 1H, J=8.6,6.0Hz), 2.03 (oct, 1H, J=6.6Hz), 0.89 (d, 3H, J=6.4Hz), 0.87 (d, 3H, J= 6.5Hz)。
(a-ii) (S) -2- (allyloxycarbonyl amino) -3 Methylbutanoic acid 2,5- dioxo pyrrolidin -1- base ester (I3)
To protection sour I2 (60.6g, 301.16mmol, 1.0 equivalent) and n-hydroxysuccinimide (34.66g, 301.16mmol, 1.0 equivalents) in the agitating solution in anhydrous THF (800mL) add dicyclohexylcarbodiimide (62.14g, 301.16mmol 1 equivalent).At room temperature by reaction stirring 18 hours.Then reaction mixture is filtered, washs solid simultaneously with THF It is concentrated under reduced pressure the filtrate of merging.Residual is re-dissolved in DCM and is allowed to rest for 30 minutes at 0 DEG C.Filter suspension And it is washed with cold DCM.Product (the 84.7g, it is assumed that 100% produces that the filtrate being concentrated under reduced pressure provides as thick colorless oil Rate), next step is used for without being further purified.LC/MS(2.194min(ES+)), m/z:321.0 [M+Na]+。1H NMR (400MHz, DMSO-d6) δ 8.0 (d, 1H, J=8.3Hz), 5.97-5.87 (m, 1H), 5.30 (ddd, 1H, J=17.2, 3.0,1.7Hz), 5.19 (ddd, 1H, J=10.4,2.7,1.4Hz), 4.52 (dt, 2H, J=5.3,1.4Hz), 4.32 (dd, 1H, J=8.3,6.6Hz), 2.81 (m, 4H), 2.18 (oct, 1H, J=6.7Hz), 1.00 (d, 6H, J=6.8Hz),
(a-iii) (S) -2- ((S) -2- (allyloxycarbonyl amino) -3- methylbutylamino) propionic acid (I4)
Solution of the succinimide ester I3 (12.99g, 43.55mmol, 1.0 equivalent) in THF (50mL) is added to L- Alanine (4.07g, 45.73mmol, 1.05 equivalent) and NaHCO3(4.02g, 47.90mmol, 1.1 equivalent) is at THF (100mL) And H2In solution in O (100mL).When removing THF under reduced pressure, stir the mixture at room temperature 72 hours.Use lemon PH is adjusted to 3-4 with precipitate white glue (gum) by acid.After ethyl acetate (6 × 150mL) extraction, H is used2O (200mL) washing Combined organic matter, uses MgSO4It dries, filters and is concentrated under reduced pressure.With diethyl ether development to be provided as the product of white powder simultaneously By filtering and using diethyl ether (5.78g, 49%) washing to collect the product.LC/MS(1.925min(ES+)), m/z:273.1 [M+H]+。1H NMR (400MHz, DMSO-d6) δ 12.47 (br s, 1H), 8.17 (d, 1H, J=6.8Hz), 7.16 (d, 1H, J= 9.0Hz), 5.95-5.85 (m, 1H), 5.29 (dd, 1H, J=17.2,1.7Hz), 5.17 (dd, 1H, J=10.4,1.5Hz), 4.46 (m, 2H), 4.18 (quin, 1H, J=7.2Hz), 3.87 (dd, 1H, J=9.0,7.1Hz), 1.95 (oct, 1H, J= 6.8Hz), 1.26 (d, 3H, J=7.3Hz), 0.88 (d, 3H, J=6.8Hz), 0.83 (d, 3H, J=6.8Hz)
(a-iv) (S)-1- ((S)-1- (4- (methylol) phenylamino)-1- oxo propyl- 2- base amino)-3- methyl-1-oxygen For butyl- 2- aminocarbamic acid allyl ester (I5)
EEDQ (5.51g, 22.29mmol, 1.05 equivalent) is added to aminobenzyl alcohol (2.74g, 22.29mmol, 1.05 Equivalent) and acid I4 (5.78g, 21.23mmol, 1 equivalent) in the solution in anhydrous THF (100mL) and be stirred at room temperature 72 Hour.Then, reaction mixture is concentrated under reduced pressure and develops resulting brown solid with diethyl ether and filters, then with excessive two Ether is washed to be provided as the product (7.1g, 88%) of pale solid.LC/MS(1.980min(ES+)), m/z:378.0 [M+ H]+。1H NMR (400MHz, DMSO-d6) δ 9.89 (br s, 1H), 8.13 (d, 1H, J=7.0Hz), 7.52 (d, 2H, J= 8.5Hz), 7.26 (m, 1H), 7.23 (d, 2H, J=8.5Hz), 5.91 (m, 1H), 5.30 (m, 1H), 5.17 (m, 1H), 4.46 (m, 2H), 5.09 (t, 1H, J=5.6Hz), 4.48 (m, 2H), 4.42 (m, 3H), 3.89 (dd, 1H, J=8.6,6.8Hz), 1.97 (m, 1H), 1.30 (d, 3H, J=7.1Hz), 0.88 (d, 3H, J=6.8Hz), 0.83 (d, 3H, J=6.7Hz).(b)
Four oxa- -3- azepine octadecane -18- of 1- iodo -2- oxo -6,9,12,15- is sour (I7)
Solution of the iodoacetic anhydride (0.250g, 0.706mmol, 1.1 equivalent) in anhydrous DCM (1mL) is added to ammonia Base-PEG(4)Acid I6 (0.170g, 0.642mmol, 1.0 equivalent) is in the solution in DCM (1mL).It stirs in the dark at room temperature Mixture is mixed to stay overnight.With 0.1M HCl, water washing reaction mixture, MgSO is used4It dries, filters and is concentrated under reduced pressure.By quick (silica gel, the solution of 3% methanol and 0.1% formic acid in chloroform to 10% methanol and 0.1% formic acid are molten in chloroform for chromatography Liquid) residue is purified to be provided as the product (0.118g, 42%) of orange oil.LC/MS(1.623min(ES+)), m/z: 433.98[M+H]+。1H NMR (400MHz, CDCl3) δ 8.069 (s, 1H), 7.22 (br s, 1H), 3.79 (t, 2H, J= 5.8Hz), 3.74 (s, 2H), 3.72-3.58 (m, 14H), 3.50-3.46 (m, 2H), 2.62 (t, 2H, J=5.8Hz).
(ii) (11S, 11aS) -11- (t-butyldimethylsilyloxy base) -8- (3- iodo propoxyl group) -7- methoxyl group - 5- oxo -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10 (5H)-carboxylic acid allyl esters (74)
(a) (S)-trifluoromethanesulfonic acid 5- ((t-butyldimethylsilyl oxygroup) methyl) -1- (5- methoxyl group -2- nitre Base -4- (triisopropyl siloxy) benzoyl) -4,5- dihydro -1H- pyrroles -3- base ester (47)
At -50 DEG C, trifluoromethanesulfanhydride anhydride (28.4g, 100.0mmol, 3.0 equivalent) is added dropwise in 25 minutes to play Ketone 46 (19.5g, 30.0mmol, 1.0 for containing 2,6- lutidines (14.4g, 130.0mmol, 4.0 equivalent) of strong stirring Equivalent) in the solution in DCM (550mL).It is stirred to react mixture 1.5 hours, LC/MS Indicator Reaction is completed at this time.Successively Organic phase is washed with water (100mL), saturated sodium bicarbonate (150mL), salt water (50mL) and uses MgSO4Dry organic phase, decompression It filters and is concentrated.By flash chromatography (silica gel, 90/10v/v n-hexane/EtOAc) purifying residual to be provided as light yellow oil Product (19.5g, 82%).LC/MS(4.391min(ES+)), m/z:713.25 [M+H]+。1H NMR (400MHz, CDCl3)δ 7.68 (s, 1H), 6.72 (s, 1H), 6.02 (t, 1H, J=1.9Hz), 4.75 (m, 1H), 4.05 (m, 2H), 3.87 (s, 3H), 3.15 (ddd, 1H, J=16.2,10.3,2.3Hz), 2.96 (ddd, 1H, J=16.2,4.0,1.6Hz), 1.28-1.21 (m, 3H), 1.07 (d, 18H, J=7.2Hz), 0.88 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H).
(b) (S, E)-(2- ((t-butyldimethylsilyl oxygroup) methyl) -4- (propyl- 1- alkenyl) -2,3- dihydro - 1H- pyrroles -1- base) (5- methoxyl group -2- nitro -4- (triisopropyl siloxy) phenyl) ketone (67)
In a nitrogen atmosphere, tetrakis triphenylphosphine palladium (0) (0.41g, 0.35mmol, 0.03 equivalent) is added to fluoroform Sulphonic acid ester 47 (8.4g, 11.8mmol, 1.0 equivalent), E-1- propylene -1- ylboronic acid (1.42g, 16.5mmol, 1.4 equivalent) and phosphorus Sour potassium (5.0g, 23.6mmol, 2.0 equivalent) is in the mixture in anhydrous dioxanes (60mL).At 25 DEG C, mixture is stirred 120 minutes, LC/MS showed fully reacting at this time.Ethyl acetate (120mL) and water (120mL) is added, removes organic phase, uses salt Water (20mL) washing, uses MgSO4It is dry, it is filtered under diminished pressure and is concentrated.Pass through flash chromatography (silica gel, 95/5v/v n-hexane/EtOAc Residual is purified to 90/10v/v n-hexane/EtOAc) to be provided as the product (4.96g, 70%) of yellow colored foam.LC/MS (4.477min(ES+)), m/z:605.0 [M+H]+。1H NMR (400MHz, CDCl3) δ 7.67 (s, 1H), 6.74 (s, 1H), (5.93 d, 1H, J=15.4Hz), 5.67 (s, 1H), 4.65 (m, 1H), 4.04 (m, 2H), 3.86 (s, 3H), 2.85 (m, 1H), 2.71 (m, 1H), 1.72 (dd, 3H, J=6.8,1.0Hz), 1.30-1.22 (m, 3H), 1.07 (d, 18H, J=7.2Hz), 0.87 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).
(c) (S, E)-(2- amino -5- methoxyl group -4- (triisopropyl siloxy) phenyl) (2- ((tert-butyl diformazan Base silicyl oxygroup) methyl) -4- (propyl- 1- alkenyl) -2,3- dihydro -1H- pyrroles -1- base) ketone (68)
Temperature is maintained between 25-30 DEG C using ice bath, in 20 minutes, by zinc powder, (22.0g, 0.33mol, 37 work as Amount) acrylic intermediate 67 (5.5g, 9.1mmol, 1.0 equivalent) is partially added in 5%v/v formic acid/ethyl alcohol (55mL) In solution.After 30 minutes, pass through the short bed filtration reaction mixture of diatomite.Diatomite is washed simultaneously with ethyl acetate (65mL) Successively the organic phase merged is washed with water (35mL), saturated sodium bicarbonate (35mL) and salt water (10mL).Use MgSO4Drying is organic Phase is filtered under diminished pressure and is concentrated.It is pale yellow to be provided as by flash chromatography (silica gel, 90/10v/v n-hexane/EtOAc) purifying residual The product (3.6g, 69.0%) of color oil.LC/MS(4.439min(ES+)), m/z:575.2 [M+H]+。1H NMR (400MHz, CDCl3) δ 6.75 (m, 1H), 6.40 (br s, 1H), 6.28 (m, 1H), 6.11 (d, 1H, J=15.4Hz), 5.53 (m, 1H), 4.67 (m, 1H), 4.36 (m, 2H), 3.93 (br s, 1H), 3.84 (br s, 1H), 3.73 (s, 3H), 2.86 (dd, 1H, J= 15.7,10.4Hz), 2.73 (dd, 1H, J=15.9,4.5Hz), 1.80 (dd, 3H, J=6.8,1.3Hz), 1.35-1.23 (m, 3H), 1.12 (d, 18H, J=7.3Hz), 0.89 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H).
(d) (S, E) -2- (2- ((t-butyldimethylsilyl oxygroup) methyl) -4- (propyl- 1- alkenyl) -2,3- two Hydrogen -1H- pyrroles -1- carbonyl) -4- methoxyl group -5- (triisopropyl siloxy) phenylcarbamic acid allyl ester (69)
At -78 DEG C, allyl chlorocarbonate (0.83g, 6.88mmol, 1.1 equivalent) is added to containing anhydrous pyridine Solution of the amine 68 (3.6g, 6.26mmol, 1.0 equivalent) of (1.09g, 13.77mmol, 2.2 equivalent) in anhydrous DCM (80mL) In.It removes dry ice and reaction mixture is warming up to room temperature.After being stirred for 15 minutes, LC/MS shows fully reacting.Successively use 0.01N HCl (50mL), saturated sodium bicarbonate (50mL), salt water (10mL) wash organic phase, use MgSO4It is dry, it is filtered under diminished pressure And be concentrated to generate light yellow oil, the oil does not have to be further purified and be used in next step (4.12g, it is assumed that yield 100%). LC/MS(4.862min(ES+)), m/z:659.2 [M+H]+。
(e) (S, E) -2- (2- (hydroxymethyl) -4- (propyl- 1- alkenyl) -2,3- dihydro -1H- pyrroles -1- carbonyl) -4- first Oxygroup -5- (triisopropyl siloxy) phenylcarbamic acid allyl ester (70)
Crude intermediate 69 (it is assumed that yield 100%, 4.12g, 6.25mmol, 1.0 equivalent) is dissolved in acetic acid (70mL), first In the mixture of alcohol (10mL), THF (10mL) and water (20mL) and it is stirred at room temperature.After 6 hours, ethyl acetate is used (500mL) diluted reaction mixture is simultaneously successively washed with water (2 × 500mL), saturated sodium bicarbonate (300mL) and salt water (50mL) It washs.Use MgSO4Dry organic phase, is filtered under diminished pressure and is concentrated.Pass through flash chromatography (silica gel, 1/99v/v methanol/DCM to 5/95v/ V methanol/DCM) residue is purified to be provided as the product of yellow oil and recycle the unreacted starting material of 1g.Make the material It is subjected to reaction condition as described above, but stirs 16h.Post-processing and after purification, separate other product (2.7g, 79%, 2 steps), LC/MS (3.742min (ES+), m/z:545.2 [M+H]+。1H NMR (400MHz, CDCl3) δ 8.38 (m, 1H), 7.72 (m, 1H), 6.81 (s, 1H), 6.37 (m, 1H), 6.10 (d, 1H, J=15.8Hz), 5.97 (m, 1H), 5.53 (m, 1H), 5.36 (ddd, 1H, J=17.2,3.1,1.5Hz), 5.25 (ddd, 1H, J=10.4,2.5,1.3Hz), 4.78 (m, 1H), 4.65 (dt, 2H, J=5.7,1.3Hz), 3.84 (m, 3H), 3.79 (s, 3H), 3.04 (dd, 1H, J=16.7,10.5Hz), 2.40 (dd, 1H, J=16.0,4.5Hz), 1.82 (dd, 3H, J=6.8,1.0Hz), 1.36-1.26 (m, 3H), 1.14 (d, 18H, J= 7.3Hz)。
(f) (11S, 11aS) -11- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -8- (triisopropyl first Siloxy) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid allyls Ester (71)
Under nitrogen atmosphere, anhydrous dimethyl sulfoxide (1.16g, 14.87mmol, 3.0 equivalent) is added dropwise to grass at -78 DEG C Acyl chlorides (0.94g, 7.43mmol, 1.5 equivalent) is in the solution in DCM (25mL).- 78 DEG C are kept the temperature at, after ten minutes, Solution of the primary alconol 70 (2.7g, 4.96mmol, 1.0 equivalent) in DCM (20mL) is added dropwise.After other 15 minutes, nothing is added Water triethylamine (2.5g, 24.78mmol, 5.0eq) and reaction mixture is heated to room temperature.Successively with the HCl of cold 0.1N (50ml), saturated sodium bicarbonate (50mL), salt water (10mL) washing reaction mixture simultaneously use MgSO4Dry organic layer, was depressurized It filters and is concentrated to be produced as the product of yellow oil, the product does not have to be further purified and be used for (2.68g, it is assumed that produce in next step Rate 100%).LC/MS(3.548min(ES+)), m/z:543.2 [M+H]+。
(g) (11S, 11aS) -11- (t-butyldimethylsilyloxy base) -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -8- (triisopropylsilyl oxygroup) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] phenodiazine It is miscellaneous10 (5H)-carboxylic acid allyl esters (72)
Under nitrogen atmosphere, at 0 DEG C, by tert-butyl dimethyl silyl triflate, (3.93g, 14.87mmol, 3.0 work as Amount) it is added to carbinolamine 71 (it is assumed that yield 100%, 2.68g, 4.96mmol, 1.0 equivalent) and 2,6- lutidines (2.12g, 19.83mmol, 4.0 equivalent) is in the solution in anhydrous DCM (40mL).After ten minutes, reaction mixture is heated To room temperature and it is further stirred for 60 minutes.It is successively washed with water (10mL), saturated sodium bicarbonate (10mL) and salt water (5mL) organic Phase uses MgSO4It is dry, it is filtered under diminished pressure and is concentrated.It is purified by flash chromatography (silica gel, chloroform to 2/98v/v alcohol/chloroform) residual Excess is to be provided as the product (2.0g, 63%, 2 step) of yellow oil.LC/MS(4.748min(ES+)), m/z:657.2 [M+H]+。1H NMR (400MHz, CDCl3) δ 7.19 (s, 1H), 6.86 (m, 1H), 6.66 (s, 1H), 6.22 (d, 1H, J=15.4Hz), 5.81 (d, 1H, J=8.8Hz), 5.78 (m, 1H), 5.48 (m, 1H), 5.11 (d, 1H, J=5.0Hz), 5.08 (m, 1H), 4.58 (dd, 1H, J=13.4,5.4Hz), 4.35 (dd, 1H, J=13.2,5.7Hz), 3.83 (s, 3H), 3.76 (s, 1H), 3.00 (dd, 1H, J=15.6,11.0Hz), 2.53 (m, 1H), 1.81 (dd, 3H, J=6.8,0.9Hz), 1.30-1.18 (m, 3H), 1.08 (d, 9H, J=2.3Hz), 1.06 (d, 9H, J=2.3Hz), 0.86 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H).
(h) (11S, 11aS) -11- (t-butyldimethylsilyl oxygroup) -8- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza- 10 (5H)-carboxylic acids Allyl ester (73)
At 25 DEG C, two hydrations lithium acetate (0.31g, 3.04mmol, 1.0eq) are added to diaza72 (2.0g, 3.04mmol, 1.0eq) in the solution in aqueous DMF (20mL) and stir 4 hours.With ethyl acetate (200mL) diluting reaction Mixture uses MgSO successively with 0.1M citric acid (50mL, pH 3), water (50mL) and salt water (10mL) washing4It is dry, decompression It filters and is concentrated.It is purified by flash chromatography (silica gel, 50/50v/v n-hexane/EtOAc to 25/75v/v n-hexane/EtOAc) Residual is to provide product as light yellow solid (0.68g, 45%).LC/MS(3.352min(ES+)), m/z:501.1 [M+H]+。1H NMR (400MHz, CDCl3) δ 7.02 (s, 1H), 6.66 (m, 1H), 6.53 (s, 1H), 6.03 (d, 1H, J=15.5Hz), 5.80 (s, 1H), 5.63 (d, 1H, J=8.9Hz), 5.55 (m, 1H), 5.29 (m, 1H), 4.87 (m, 2H), 4.39 (dd, 1H, J= 13.5,4.2Hz), 4.20 (dd, 1H, J=13.2,5.7Hz), 3.73 (s, 3H), 3.59 (m, 1H), 2.81 (dd, 1H, J= 16.1,10.5Hz), 2.35 (d, 1H, J=15.7Hz), 1.61 (d, 3H, J=6.4Hz), 0.67 (s, 9H), 0.05 (s, 3H), 0.00 (s, 3H).
(i) (11S, 11aS) -11- (t-butyldimethylsilyl oxygroup) -8- (3- iodo propoxyl group) -7- methoxy Base -5- oxo -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10 (5H)-carboxylic acid allyl esters (74)
By diiodo propane (0.295g, 1.00mmol, 5.0 equivalent) and potassium carbonate (0.028g, 0.20mmol, 1.0 equivalent) Phenol 33 (0.100g, 0.020mmol, 1.0 equivalent) is added in the solution in dry acetone (5ml).By reaction mixture It is heated 6 hours at 60 DEG C, LC/MS shows fully reacting at this time.Reaction mixture is concentrated under reduced pressure to drying and by fast Fast chromatography (silica gel, 75/25v/v n-hexane/EtOAc to 50/50v/v n-hexane/EtOAc) purification residues are colourless to be provided as The product (0.074g, 56%) of oil.LC/MS(3.853min(ES+)), m/z:669.0 [M+H]+。1H NMR (400MHz, CDCl3) δ 7.26 (s, 1H), 6.90 (s, 1H), 6.68 (s, 1H), 6.24 (d, 1H, J=15.3Hz), 5.87 (d, 1H, J= 8.9Hz), 5.78 (m, 1H), 5.53 (m, 1H), 5.12 (m, 2H), 4.65 (m, 2H), 4.41 (m, 1H), 4.11 (m, 1H), 3.93 (s, 3H), 3.81 (m, 1H), 3.40 (t, 2H, J=6.7Hz), 3.05 (dd, 1H, J=16.3,10.1Hz), 2.57 (m, 1H), 2.34 (m, 2H), 1.84 (d, 3H, J=6.6Hz), 0.92 (s, 9H), 0.28 (s, 3H), 0.26 (s, 3H).
(iii) (11S, 11aS) -11- (t-butyldimethylsilyloxy base) -8- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza - 10 (5H)-carboxylic acids 4- ((S) -2- ((S) -2- (allyloxy carbonyl ammonia base) -3- methylbutylamino) the third amino) benzyl ester (79)
(a) ((S) -1- (((S) -1- ((4- ((((2- ((S) -2- (((t-butyldimethylsilyl) oxygroup) first Base) -4- ((E) -propyl- 1- alkene -1- base) -2,3- dihydro -1H- pyrroles -1- carbonyl) -4- methoxyl group -5- ((triisopropyl monosilane Base) oxygroup) phenyl) carbamoyl) oxygroup) methyl) phenyl) amino)-1- oxopropan-2- base) amino)-3- methyl-1- Oxo-butanes -2- base) allyl carbamate (75)
Under 5 DEG C (ice bath), by triethylamine (0.256ml, 1.84mmol, 2.2 equivalent) be added to amine 68 (0.480g, 0.835mmol, 1.0 equivalents) and stirring of the triphosgene (0.089g, 0.301mmol, 0.36 equivalent) in anhydrous THF (15mL) In solution.It is anti-to monitor isocyanates by removing part from reaction mixture interval, being terminated with methanol and carrying out lcms analysis The progress answered.Once the reaction of isocyanates is completed, then by injection by alloc-Val-Ala-PABOH I5 (0.473g, 1.25mmol, 1.5 equivalents) and solution of the triethylamine (0.174ml, 1.25mmol, 1.5 equivalent) in anhydrous THF (10mL) it is fast Speed is added in freshly prepared isocyanates.At 40 DEG C, reaction stirring 4 hours is then stirred at room temperature overnight.Decompression Be concentrated mixture and by flash chromatography (silica gel, 20/80v/v n-hexane/EtOAc to 50/50v/v n-hexane/EtOAc, then 1/99v/v DCM/MeOH to 5/95v/v DCM/MeOH) it purifies to provide yellow solid product (0.579g, 71%).LC/ MS(4.468min(ES+)), m/z:978.55 [M+H]+。1H NMR (400MHz, CDCl3) δ 8.63 (br s, 1H), 8.42 (s, 1H), 7.78 (br s, 1H), 7.53 (d, 2H, J=8.1Hz), 7.31 (d, 2H, J=8.6Hz), 6.76 (s, 1H), 6.59 (d, 1H, J=7.6Hz), 6.36 (br s, 1H), 6.04 (d, 1H, J=15.9Hz), 5.90 (m, 1H), 5.55 (m, 1H), 5.33- 5.21 (m, 3H), 5.10 (s, 2H), 4.66 (m, 2H), 4.57 (dd, 2H, J=5.6,1.0Hz), 3.98 (dd, 1H, J=7.3, 6.8Hz), 3.90 (m, 1H), 3.81 (m, 1H), 3.78 (s, 3H), 2.82 (dd, 1H, J=15.4,9.6Hz), 2.72 (dd, 1H, J=15.9,3.5Hz), 2.17 (m, 1H), 1.78 (dd, 3H, J=6.5,0.8Hz), 1.46 (d, 3H, J=7.1Hz), 1.29 (m, 3H), 1.11 (d, 18H, J=7.1Hz), 0.97 (d, 3H, J=6.8Hz), 0.92 (d, 3H, J=6.8Hz), 0.83 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H).
(b) ((S) -1- (((S) -1- ((4- ((((2- ((S) -2- (methylol) -4- ((E) -propyl- 1- alkene -1- base) -2,3- Dihydro -1H- pyrroles -1- carbonyl) -4- methoxyl group -5- ((triisopropylsilyl) oxygroup) phenyl) carbamoyl) oxygroup) Methyl) phenyl) amino)-1- oxopropan-2- base) amino)-3- methyl-1-oxo-butanes-2- base) allyl carbamate (76)
By silyl ether 75 (1.49g, 1.52mmol, 1.0 equivalent) be dissolved in acetic acid/methanol/tetrahydrofuran/water (14: In 7:1:1:2 mixture 2:2:4ml) and it is stirred at room temperature.After 2 hours, with EtOAc (100mL) diluting reaction, successively use Water, sodium bicarbonate aqueous solution washing, are then washed with brine.Then MgSO is used4Dry organic phase, is filtered under diminished pressure and is concentrated.It is logical Flash chromatography (silica gel, 100/0, then 99/1 to 92/8v/v DCM/ methanol) purifying residue is crossed to be provided as orange solids Product (1.2g, 92%).LC/MS(3.649min(ES+)), m/z:865.44 [M+H]+。1H NMR (400MHz, CDCl3)δ 8.44 (s, 1H), 8.35 (s, 1H), 7.69 (br s, 1H), 7.53 (d, 2H, J=8.7Hz), 7.32 (d, 2H, J=8.3Hz), 6.78 (s, 1H), 6.56 (m, 2H), 6.32 (br s, 1H), 6.05 (d, 1H, J=14.9Hz), 5.90 (m, 1H), 5.56 (m, 1H), 5.30 (m, 2H), 5.22 (m, 1H), 5.10 (d, 2H, J=3.1Hz), 4.73 (m, 1H), 4.64 (m, 1H), 4.57 (d, 2H, J=5.8Hz), 4.01 (m, 1H), 3.79 (m, 2H), 3.76 (s, 3H), 2.98 (dd, 1H, J=16.3,10.2Hz), 2.38 (dd, 1H, J=16.6,4.1Hz), 2.16 (m, 1H), 1.78 (dd, 3H, J=6.8,0.9Hz), 1.46 (d, 3H, J= 7.1Hz), 1.29 (m, 3H), 1.11 (d, 18H, J=7.4Hz), 0.97 (d, 3H, J=6.7Hz), 0.92 (d, 3H, J= 6.8Hz)。
(c) (11S, 11aS) -11- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -8- (triisopropyl first Siloxy) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid 4- ((S)- 2- ((S) -2- (allyloxy carbonyl ammonia base) -3- methylbutylamino) third amino) benzyl ester (77)
Under nitrogen atmosphere, anhydrous dimethyl sulfoxide (0.180g, 2.3mmol, 3.0 equivalent) is added dropwise to grass at -78 DEG C Acyl chlorides (0.147g, 1.1mmol, 1.5 equivalent) is in the solution in DCM (10mL).Temperature is maintained -78 DEG C, after twenty minutes, Solution of the primary alconol 76 (0.666g, 0.77mmol, 1.0 equivalent) in DCM (10mL) is added dropwise.After other 15 minutes, it is added Anhydrous triethylamine (0.390g, 3.85mmol, 5.0 equivalent) and reaction mixture is warming up to room temperature.Successively with cold 0.1N HCl (10mL), saturated sodium bicarbonate (10mL) and salt water (5mL) washing reaction mixture.Then MgSO is used4Drying is organic Layer, is filtered under diminished pressure and is concentrated.Then by flash chromatography (silica gel, 50/50v/v n-hexane/EtOAc to 25/75v/v n-hexane/ EtOAc) purifying residual is to provide white solid product (0.356g, 54%).LC/MS(3.487min(ES+)), m/z: 862.2[M+H]+。1H NMR (400MHz, CDCl3) δ 8.34 (br s, 1H), 7.47 (d, 2H, J=7.6Hz), 7.17 (s, 1H), 7.14 (d, 2H, J=7.5Hz), 6.86 (br s, 1H), 6.65 (br s, 1H), 6.42 (d, 1H, J=7.6Hz), 6.22 (d, 1H, J=14.4Hz), 5.80 (m, 1H), 5.40 (m, 1H), 5.53 (m, 1H), 5.32 (m, 1H), 5.21 (d, 2H, J= 9.6Hz), 5.06 (d, 1H, J=12.3Hz), 4.90 (m, 1H), 4.58 (m, 3H), 3.98 (m, 1H), 3.84 (m, 1H), 3.81 (s, 3H), 3.50 (m, 1H), 3.05 (dd, 1H, J=16.0,10.3Hz), 2.76 (m, 1H), 2.15 (m, 1H), 1.80 (dd, 3H, J=6.7,0.8Hz), 1.44 (d, 3H, J=7.1Hz), 1.16 (m, 3H), 1.01 (d, 18H, J=6.6Hz), 0.96 (d, 3H, J=6.8Hz), 0.92 (d, 3H, J=6.8Hz).
(d) (11S, 11aS) -11- (t-butyldimethylsilyloxy base) -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -8- (triisopropyl siloxy) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid 4- ((S) -2- ((S) -2- (allyloxy carbonyl ammonia base) -3- methylbutylamino) the third amino) benzyl ester (78)
Under nitrogen atmosphere, at 0 DEG C, by t-butyldimethylsilyl triflate (0.46g, 1.74mmol, 3.0 equivalents) it is added to secondary alcohol 77 (0.5g, 0.58mmol, 1.0 equivalent) and 2,6- lutidines (0.25g, 2.32mmol, 4.0 equivalents) in the solution in anhydrous DCM (10mL).After ten minutes, reaction mixture is heated to room temperature and be further stirred for 120 minutes.Then organic phase successively is washed with water (10mL), saturated sodium bicarbonate (10mL) and salt water (5mL), uses MgSO4It is dry It is dry, it is filtered under diminished pressure and is concentrated.By flash chromatography (silica gel, 50/50v/v n-hexane/EtOAc) purifying residual to be provided as white The product (0.320g, 57%) of solid.LC/MS(4.415min(ES+)), m/z:976.52 [M+H]+。1H NMR (400MHz, CDCl3) δ 8.31 (br s, 1H), 7.48 (d, 2H, J=8.0Hz), 7.21 (s, 1H), 7.14 (d, 2H, J=8.3Hz), 6.89 (s, 1H), 6.65 (s, 1H), 6.38 (d, 1H, J=7.3Hz), 6.25 (d, 1H, J=14.6Hz), 5.93 (m, 1H), 5.85 (d, 1H, J=8.8Hz), 5.50 (m, 1H), 5.34 (m, 1H), 5.24 (m, 2H), 5.15 (d, 1H, J=12.5Hz), 4.86 (d, 1H, J=12.2Hz), 4.62 (m, 3H), 4.01 (m, 1H), 3.86 (s, 3H), 3.78 (m, 1H), 3.04 (m, 1H), 2.56 (m, 1H), 2.20 (m, 1H), 1.84 (dd, 3H, J=6.6,0.7Hz), 1.48 (d, 3H, J=6.8Hz), 1.20 (m, 3H), 1.05 (d, 9H, J=2.9Hz), 1.03 (d, 9H, J=2.9Hz), 0.99 (d, 3H, J=6.8Hz), 0.95 (d, 3H, J=6.8Hz), 0.88 (s, 9H), 0.27 (s, 3H), 0.14 (s, 3H).
(e) (11S, 11aS) -11- (t-butyldimethylsilyl oxygroup) -8- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza- 10 (5H)-carboxylic acids 4- ((S) -2- ((S) -2- (allyloxy carbonyl ammonia base) -3- methylbutylamino) the third amino) benzyl ester (79)
At 25 DEG C, two hydrations lithium acetate (0.010g, 0.10mmol, 1.0 equivalent) are added to silyl ether 78 (0.100g, 0.10mmol, 1.0 equivalent) is 3 hours in the solution in aqueous DMF (2mL).Then dilute with ethyl acetate (20mL) Reaction mixture is released, successively with 0.1M citric acid (20mL, pH 3), water (20mL) and salt water (5mL) washing, uses MgSO4It is dry, It is filtered under diminished pressure and is concentrated.By flash chromatography (silica gel, 5/95v/v methanol/DCM) purification residues to be provided as light yellow oil Product (0.070g, 83%).LC/MS(3.362min(ES+)), m/z:820.2 [M+H]+。1H NMR (400MHz, CDCl3)δ 8.39 (s, 1H), 7.48 (d, 2H, J=8.2Hz), 7.25 (s, 1H), 7.12 (d, 2H, J=8.1Hz), 6.88 (s, 1H), 6.68 (s, 1H), 6.47 (d, 1H, J=7.6Hz), 6.24 (d, 1H, J=15.2Hz), 6.03 (s, 1H), 5.92 (m, 1H), 5.84 (d, 1H, J=8.9Hz), 5.50 (m, 1H), 5.34 (m, 1H), 5.26 (m, 2H), 5.18 (d, 1H, J=12.3Hz), 4.80 (d, 1H, J=12.4Hz), 4.66-4.60 (m, 3H), 4.02 (m, 1H), 3.95 (s, 3H), 3.81 (m, 1H), 3.03 (m, 1H), 2.57 (m, 1H), 2.19 (m, 1H), 1.84 (dd, 3H, J=6.8,0.8Hz), 1.48 (d, 3H, J=7.1Hz), 1.00 (d, 3H, J= 6.8Hz), 0.95 (d, 3H, J=6.8Hz), 0.87 (s, 9H), 0.26 (s, 3H), 0.12 (s, 3H).
(iv) (11S, 11aS) -11- hydroxyl -7- methoxyl group -8- (3- ((S) -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -5,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 8- base oxygroup) propoxyl group) -5- oxygen Generation -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 10(5H)- Carboxylic acid 4- (four oxa- -3 (20S, 23S) -1- iodo -20- isopropyl -23- methyl -2,18,21- trioxy- -6,9,12,15-, 19,22- tri- azepine lignocerane amino) benzyl ester (66, D)
(a) (11S, 11aS) -8- (3- ((11S, 11aS) -10- ((4- ((R) -2- ((R) -2- (allyloxy carbonyl ammonia Base) -3- methylbutylamino) the third amino) benzyloxy) carbonyl) -11- (t-butyldimethylsilyloxy base) -7- methoxyl group -5- Oxo -2- ((E) -propyl- 1- alkenyl) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza 8- base oxygroup) propoxyl group) -11- (t-butyldimethylsilyloxy base) -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkene Base) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid allyl esters (80)
Potassium carbonate (0.030g, 0.21mmol, 1.0 equivalent) is added to phenol 79, and (0.175g, 0.21mmol, 1.0 work as Amount) and iodine connector 74 (0.214g, 0.32mmol, 1.5 equivalent) in the solution in acetone (10mL).By reaction mixture in nitrogen It is heated 17 hours in sealing triangular flask under atmosphere at 75 DEG C.Reaction mixture is concentrated under reduced pressure to drying and by quick Chromatography (silica gel, 2/98v/v methanol/DCM to 5/95v/v methanol/DCM) is purified to be provided as the product of light yellow solid (0.100g, 35%).LC/MS(4.293min(ES+)), m/z:1359.13 [M]+。
(b) (11S, 11aS) -8- (3- ((11S, 11aS) -10- ((4- ((R) -2- ((R) -2- (allyloxy carbonyl ammonia Base) -3- methylbutylamino) the third amino) benzyloxy) carbonyl) -11- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkene Base) -5,10,11,11a- tetrahydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- base oxygroup) propoxyl group)- 11- hydroxyl -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10 (5H)-carboxylic acid allyl esters (81)
By four n-butylamine fluorides (1M, 0.22mL, 0.22mmol, 2 equivalent) be added to silyl ether 80 (0.150g, 0.11mmol, 1.0 equivalents) in the solution in anhydrous THF (2mL).Reaction mixture is stirred at room temperature 20 minutes, at this time LC/MS Indicator Reaction is complete.It is sequentially washed with ethyl acetate (10mL) diluted reaction mixture and with water (5mL) and salt water (5mL) It washs.Use MgSO4Dry organic phase, is filtered under diminished pressure and is concentrated to leave yellow solid.Pass through flash chromatography (silica gel, 6/94v/v first Alcohol/DCM to 10/90v/v methanol/DCM) it is purified to be provided as the product of light yellow solid (0.090g, 73%).LC/MS (2.947min(ES+)), m/z:1154.0 [M+Na]+。1H NMR (400MHz, CDCl3) δ 8.39 (br s, 1H), 7.39 (d, 2H, J=7.6Hz), 7.18 (d, 2H, J=10.6Hz), 7.10 (m, 3H), 6.86 (d, 2H, J=10.0Hz), 6.74 (s, 1H), 6.55 (s, 1H), 6.22 (dd, 2H, J=15.3,6.6Hz), 5.85 (m, 2H), 5.74 (m, 3H), 5.52 (m, 2H), 5.22 (m, 1H), 5.00 (m, 2H), 4.57 (m, 6H), 4.41 (m, 2H), 4.09 (m, 4H), 3.85 (m, 11H), 3.06 (m, 2H), 2.76 (m, 2H), 2.20 (m, 2H), 2.08 (m, 1H), 1.79 (d, 6H, J=6.4Hz), 1.40 (d, 3H, J=6.1Hz), 0.90 (m, 6H)。
(c) (11S, 11aS) -11- hydroxyl -7- methoxyl group -8- (3- ((S) -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- Alkenyl) -5,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- base oxygroup) propoxyl group) -5- oxygen Generation -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10(5H)- Carboxylic acid 4- ((R) -2- ((R) -2- amino -3- methylbutylamino) the third amino) benzyl ester (65)
Tetrakis triphenylphosphine palladium (0) (0.005g, 0.005mmol, 0.06 equivalent) is added to double-carbinolamine 81 (0.090g, 0.08mmol, 1.0 equivalent) and pyrrolidines (16 μ L, 0.20mmol, 2.5 equivalent) are molten in anhydrous DCM (5mL) In liquid.After twenty minutes, with DCM (10mL) diluted reaction mixture and successively with saturated aqueous ammonium chloride (5mL) and salt water (5mL) washing, uses MgSO4It is dry, it is filtered under diminished pressure and removes solvent with remaining pale-yellow solid crude product, without being further purified It is used for next step (0.075g, it is assumed that 100% yield).LC/MS(2.060min(ES+)), m/z:947.2 [M+H]+。
(d) (11S, 11aS) -11- hydroxyl -7- methoxyl group -8- (3- ((S) -7- methoxyl group -5- oxo -2- ((E) -propyl- 1- Alkenyl) -5,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza8- base oxygroup) propoxyl group) -5- oxygen Generation -2- ((E) -propyl- 1- alkenyl) -11,11a- dihydro -1H- benzo [e] pyrrolo- [1,2-a] [1,4] diaza10(5H)- Carboxylic acid 4- (four oxa- -3 (20S, 23S) -1- iodo -20- isopropyl -23- methyl -2,18,21- trioxy- -6,9,12,15-, 19,22- tri- azepine lignocerane amino) benzyl ester (66, D)
By EDCI (0.015g, 0.08mmol, 1.0 equivalent) be added to amine 65 (it is assumed that 100% yield, 0.075g, 0.08mmol, 1.0 equivalents) and iodo-acetamide-PEG4Acid I7 (0.034g, 0.08mmol, 1.0 equivalent) is in anhydrous dichloromethane It is stirred to react in solution in alkane (5mL) and in the dark.After 50 minutes, the iodo-acetamide-EG of additional amount is added4Acid I7 The EDCI (0.003g, 0.016mmol, 0.2 equivalent) of (0.007g, 0.016mmol, 0.2 equivalent) and additional amount.2.5 is small in total Shi Hou with methylene chloride (15mL) diluted reaction mixture and is successively washed with water (10mL) and salt water (10mL).Use MgSO4It is dry Dry organic phase is filtered under diminished pressure and is concentrated.It is purified by flash chromatography (silica gel, chloroform 100% to 90:10v/v chloroform: methanol) The residue arrived.Merge pure part to provide product (0.0254g, 23%, 2 step).It collects crude product part and passes through preparation Type TLC (silica gel, 90:10v/v chloroform: methanol) is purified to provide the product (0.0036g, 3%, 2 step) of second batch.LC/MS (2.689min(ES+)), m/z:681.01/2 [M+2H]+。
Embodiment 10: the activity of the compound of release
K562 measurement
At 37 DEG C, containing 5%CO2Humid atmosphere in, K562 human chronic's marrow leukaemia cell is maintained at supplement Have in the RPM11640 culture medium of 10% fetal calf serum and 2mM glutamine and 37 DEG C in the dark, with the medicine of given dose Object is incubated for 1 hour or 96 hours.It is terminated by centrifugation (5min, 300g) and is incubated for and washs cell one with the culture medium of no drug It is secondary.After drug-treated appropriate, cell is transferred to (10 in the microtiter plate in the hole 96-4Cells/well, 8 holes/sample).It will The plate is maintained at 37 DEG C, containing 5%CO2Humid atmosphere dark place.Measurement is with living cells by yellow soluble tetrazolium salts (bromination 3- (4,5- dimethylthiazole -2- base) -2,5- diphenyl -2H- tetrazolium (MTT, Aldrich-Sigma)) it is reduced to insoluble purple Based on the ability of Se formazan precipitating.Next plate is incubated for 4 days (to allow to control cell in quantitative aspects increase about 10 Times), plate to each hole and is further incubated for 5h by 20 μ L MTT solution (5mg/mL in the physiological saline of phosphate-buffered).So Plate 5min is centrifuged with 300g afterwards, most of culture mediums are sucked out from cell precipitation, leave 10-20 holes μ L/.It is added into every hole DMSO (200 μ L) and stirred sample are to ensure to be thoroughly mixed.Then in Titertek Multiscan ELISA microplate reader Optical density is read under the wavelength of 550nm and constructs dose-response curve.For every curve, by IC50Value is read as will be final Dosage needed for optical density is reduced to the 50% of control value.
In the measurement, compound R elC has the IC less than 0.1pM50。
In the measurement, compound R elE has the IC of 0.425nM50。
Embodiment 11: the formation of conjugate
General antibody binding step
In reduction buffer solution, (example: phosphate buffered saline PBS, histidine buffer solution, sodium borate buffer are molten Liquid, TRIS buffer solution) in antibody is diluted to 1-5mg/mL.By freshly prepared TCEP (three (2- carboxyethyl) phosphonium salt hydrochlorates) Solution be added in the cysteine disulphide bridges of selective reduction.The amount of TCEP and reduction (reducing, reduction) Targeting is horizontal proportional, in 1 to 4 molar equivalent/antibody, generates 2 to 8 reactive mercaptan.After 37 DEG C of reduction a few hours, Mixture is cooled to room temperature and the agent-linker of excessive addition (A, B, C, D, E) is (final as diluted DMSO solution DMSO content is up to 10% volume/reaction mixture volume).Mixture reasonable time is jiggled at 4 DEG C or at room temperature, Elongated is 1-3 hours.It is combining finally, excess reactivity mercaptan can be with " mercaptan capping reagent " (such as N- ethyl maleimide Amine (NEM)) reaction.It is combined using the centrifugal filter concentrated antibody with 10kDa or higher Molecular weight cut-off value-drug Then object is purified by tangential flow filtration (TFF) or fast protein liquid chromatogram (FPLC).Using with UV- visible light, fluorescence Or reverse chromatograms (RP) or hydrophobic interaction chromatography (HIC) that mass spectrograph detection combines, high performance liquid chromatography (HPLC) can be passed through Or ultra performance liquid chromatography (UHPLC) analysis determines corresponding antibody-drug conjugates to assess drug/Antibody ratio (DAR)； The size exclusion combined is detected using with UV- visible light, fluorescence or mass spectrograph, HPLC or UHPLC analysis aggregation water can be passed through Gentle monomer purity.It can be determined by the combination of spectrum (being absorbed at 280,214 and 330nm) and biochemical measurement final Conjugate concentration (bicinchonic acid assay BCA；Smith,P.K.,et al.(1985) Anal.Biochem.150(1):76–85；Use the IgG antibody of known concentration as reference).Usually used under aseptic condition 0.2 μm of Filter Sterile filters antibody-drug conjugates and stores it in+4 DEG C, -20 DEG C or -80 DEG C.
DAR is determined
Original antibody is gone back by 10 μ L borate buffer solutions (100mM, pH 8.4) of addition and 5 μ L DTT (0.5M in water) Or ADC (35 μ L in about 35 μ g) and 37 DEG C heat 15 minutes.With the acetonitrile of 1 volume: water: formic acid (49%:49%:2%v/v) Dilute sample and in being injected to UPLC system (Shimadzu Nexera), 80 DEG C of 3.6 μ XB- of Widepore In C18150 × 2.1mm (P/N00F-4482-AN) column (Phenomenex Aeris), flow rate 1ml/min, with 75% Buffer solution A (water, trifluoroacetic acid (0.1%v/v) (TFA), 25% buffer solution B (acetonitrile: water: TFA90%:10%:0.1% V/v it) balances.25% to 55% buffer solution gradient elution bond material is used in 10min.The UV at 214nm is integrated to absorb Peak.Next peak identified to each ADC or antibody: natural antibody light chain (L0), natural antibody heavy chain (H0) and being added with medicine Each of these chains of object-connector are (for the light chain with a drug labeled as L1 and for having 1,2 or 3 company The heavy chain of the agent-linker connect is labeled as H1, H2, H3.It for identification include the piece of agent-linker by the UV chromatography at 330nm Section (that is, L1, H1, H2, H3).
Calculate the PBD/ protein molar ratios of light chain and heavy chain:
Final DAR is calculated according to following:
DAR measurement is carried out at 214nm, because of the minimum interference that 214nm absorbs agent-linker.
Generate the ADC for being directed to PSMA
By antibody A (including variable domains, be the SEQ ID NO.3 pairs of with SEQ ID NO.4) and drug connector A In conjunction with being 2.62 with the DAR for obtaining Conj A-A and measuring.
By antibody A (including variable domains, be the SEQ ID NO.3 pairs of with SEQ ID NO.4) and drug connector B In conjunction with being 2.43 with the DAR for obtaining Conj A-B and measuring.
By antibody A (including variable domains, be the SEQ ID NO.3 pairs of with SEQ ID NO.4) and drug connector D In conjunction with being 2.35 with the DAR for obtaining Conj A-D and measuring.
By antibody A (including variable domains, be the SEQ ID NO.3 pairs of with SEQ ID NO.4) and drug connector E In conjunction with being 2.34 with the DAR for obtaining Conj A-E and measuring.
It is described below other ADC.
DeJ591 is comprising with the VH structural domain according to the sequence of SEQ ID NO.3 and with according to SEQ ID NO.4 Sequence VL structural domain anti-PSMA antibody.
By the way that deJ591 antibody is bound to the ADC that drug connector A, E or D generate targeting PSMA.Obtained ADC is listed in down In table, it is next to the DAR measured.The non-PSMA targeting ADC of control is generated using B12 AntiHIV1 RT activity gp120 antibody.
ADC DAR Concentration [mg/ml] Yield [%]
deJ591-A 2.35 2.72 66
deJ591-E 2.76 2.65 72
deJ591-D 2.0 1.42 59
B12-A 2.06 1.47 49
B12-E 1.88 1.34 68
B12-D 2.26 1.17 48
The vitro cytotoxicity of embodiment 12:ADC
Cell culture
Generous donation of the LNCaP and PC3 cell from John Hartley professor (UCL).Cell culture medium is supplement There are Pidolidone and the RPMI 1640 of 10%FBS.Cell is grown in 37 DEG C, 5%CO2, in moist incubator.
Cytotoxicity detection
Suspended cell culture (at most 1 × 10 is determined by expecting that (Trypan) indigo plant 1:1 is mixed with platform6/ ml) concentration and Survival ability and with hemacytometer rolling counters forward transparent (work)/indigo plant (dead) cell.Cell suspending liquid is diluted to required Inoculum density (usually 105/ ml) and distributed into 96 hole flat undersides.Alma indigo plant is measured, by 100 holes μ l/ point It fits in black holes plate.MTS is measured, by the distribution of 50 holes μ l/ in transparent orifice plate.Pass through the ADC dilution that will be sterile filtered The stock solution (1ml) of ADC (20 μ g/ml) is prepared into cell culture medium.By the cell that 100 μ l are continuously transferred to 900 μ l In culture medium, one group of 8 × 10 times of dilution of preparation deposit ADC in 24 orifice plates.By each ADC dilution (for alma Blue 100 holes μ l/, for 50 hole μ l/ MTS) it distributes into 4 repeating holes of 96 orifice plates comprising cell suspending liquid.Control wells are only Receive the culture medium of same volume.After being incubated for 4 days, pass through alma indigo plant or MTS analysis measurement cell viability.
It will(Invitrogen, catalog number DAL1025) distributes into each hole (20 hole μ l/) simultaneously At 37 DEG C in supply CO2Couveuse in be incubated for 4 hours.Go out measured hole fluorescence in excitation 570nm, transmitting 585nm., by 4 ADC processing hole in mean fluorecence compared with the mean fluorecence in 4 control wells (100%) ratio calculation cell survival rate (%).
MTS (Promega, catalog number G5421) is distributed into each hole to (20 hole μ l/) and at 37 DEG C in supply CO2 Couveuse in be incubated for 4 hours.It measures and absorbs at 490nm.The average absorption and 4 control wells in hole handled by 4 ADC In average absorption compared to (100%) calculate cell survival rate (%).The average data for repeating experiment by 3 groups generates dose response Curve and the dosage that data are fitted to S-shaped having a variable slope by using Prism (GraphPad, San Diego, CA) Response curve determines EC50。
Vitro cytotoxicity
The above deJ591ADC is tested to the effect of LNCaP cell.Use PC3 cell as PSMA negative control.
Fig. 1 shows vitro efficacy of the deJ591ADC in LNCaP cell.What it is with LNCap cell incubation deJ591ADC is 10 times of dilutions of column (μ g/mL).The measurement of alma indigo plant is finally carried out in the incubation stage and calculates cell survival rate.Image represents three A average value for repeating experiment.
All three deJ591ADC of test show the significant cytotoxicity (Fig. 1) to LNCaP cell.
deJ591-A deJ591-E deJ591-D
EC50μg/mL 0.01856 0.006856 0.004897
Identical experiment is repeated with PSMA-ve cell (PC3), is shown without the cytotoxic effect (Fig. 2) of concentration dependant.
The AntiHIV1 RT activity gp120 antibody (B12) that identical head (warhead) will be connected to is used as negative control ADC.Such as with On, with 10 times of dilutions of series (μ g/mL) of LNCap cell incubation B12ADC.Alma indigo plant survey is finally carried out in the incubation stage Determine and calculates cell survival rate.Image in Fig. 3 and Fig. 4 indicates the average value of three repetition experiments.
Three kinds of non-binding B12ADC conjugates show the substantially less than effect of the deJ591 of equal value in conjunction with A, E and D respectively. Identical experiment is repeated with PSMA-ve cell (PC3), is shown without the cytotoxic effect (Fig. 4) of concentration dependant again.
The internal anti-tumor activity of embodiment 13-ADC
Using the measurement of cell line LNCaP derived from PSMA+ (ve) human prostate cancer deJ591-A ADC in murine heterotropic In vivo efficacy in transplantation model.The AntiHIV1 RT activity gp120 antibody (B12) for being connected to identical drug connector is combined as non-PSMA Control.
Researching and designing
Drug and treatment:
Group number Animal/group ADC Dosage level (mg/kg) Dose volume (ml/kg)
1 10 [only carrier] - -
2 10 deJ591-A 0.3 10
3 10 deJ591-A 1.0 10
4 10 B12-A 0.3 10
5 10 B12-A 1.0 10
Step:
CR female NCr nu/nu mouse is set, there is the I x I at side of body portion (flank) in 0%Matrigel sc LNCaP-SPN tumour cell.
Cell infusion volume is 0.1mL/ mouse.
The age of from date: 8 to 12 weeks.
When tumour reaches 100-150mm3Average-size when, matched in pairs and start to treat.
Weight: qd × 5, then bi-wk is to the end.
Calliper to measure: bi-wk is to the end.
Immediately any adverse reaction or death are reported.
Be euthanized have single observation to > 30% weight loss or three times continuously measure > 25% weight loss Any individual animals.
Any group for measuring the average weight mitigation or > 10% death rate with > 20% twice can be stopped being administered. The group that is not euthanized simultaneously allows to restore.In the group with > 20% weight saving, euthanasia is reached into single weight loss terminal Individual.If treatment group relevant to weight loss restores to the 10% of original weight, can with relatively low-dose or The dosage regimen of lower frequency continues to be administered.The exception that non-treatment weight % can be allowed to restore on the basis of case analysis (exception)。
Terminal TGD.Animal will be separately monitored.Experimental endpoints are for 2000mm3Gross tumor volume or 60 days, whichever First reach.Respondent can carry out more long.When reaching terminal, be euthanized animal.
Universal method approach
Calculating for group mean tumour volume, application are regular once:, will when animal is withdrawn from the study due to tumor size Final gross tumor volume for animal record is included with data, the average external volume for calculated for subsequent time point.Error rod figure Indicate average standard error (SEM).When being less than the reservation of 50% animal in group under study for action, not by gross tumor volume value based on Calculation group mean tumour volume.It is analyzed using Prism (GraphPad, San Diego, CA) for graphical representation and data.
As a result
Fig. 5 shows the deJ591-A in LNCaP heteroplastic transplantation model.When every experimental group mean tumour volume reaches 0.1cm3When, it is administered to mouse and they is treated by the IV in tail vein with the ADC single dose of 0.3 and 1mg/kg.Number According to mean tumour volume (+/- SEM) of the expression from every group of 10 mouse.
It is active (Fig. 5) that the deJ591-A of each concentration shows effective antitumour.DeJ591-A is shown noticeably greater than The anti-tumor activity of non-binding ADC (B12-A) control.
Abbreviation
Ac acetyl group
Acm acetylamino methyl
Alloc allyloxy carbonyl
Bis- dimethyl dicarbonate butyl ester of Boc
T-Bu tert-butyl
Bzl benzyl, wherein Bzl-OMe is methoxy-benzyl and Bzl-Me is methylbenzyl
Cbz or Z benzyloxy-carbonyl, wherein Z-Cl and Z-Br is chlorobenzyloxycarbonyl and bromo-benzyloxy carbonyl respectively
DMF N,N-dimethylformamide
Dnp dinitrophenyl
DTT dithiothreitol (DTT)
Fmoc 9H- fluorenes-ylmeth-oxycarbonyl
Imp N-10 imines blocking group: 3- (2- methoxy ethoxy) propionic ester-Val-Ala-PAB
MC-OSu maleimidocaproyl-O-N- succinimide
Moc methoxycarbonyl
MP maleimide propionamide
Mtr 4- methoxyl group -2,3,6- trimethylphenysulfonyl
PAB is to aminobenzyloxycarbonyl
PEG ethyleneoxy
PNZ is to nitrobenzylamino formic acid esters
Psec 2- (phenyl sulfonyl) ethoxy carbonyl
TBDMS t-butyldimethylsilyl
TBDPS t-butyldiphenylsilyl
Teoc 2- (trimethyl silyl) ethoxy carbonyl
Tos tosyl
Troc 2,2,2- tri-chloroethoxy base carbonyl chloride
Trt trityl
Xan xanthyl

Claims (14)

1. a kind of conjugate of following formula:
ConjA:
,
ConjD:
,
Or ConjE:
Wherein, Ab is the antibody for being bound to PSMA, and the antibody includes to have according to SEQ ID NO. 1,3,5,7,8,9 or 10 In any sequence VH structural domain；And
Wherein, the drugloading rate (p) of drug (D) to antibody (Ab) is 1 to 8 integer.
2. conjugate according to claim 1, wherein the antibody further include have according to SEQ ID NO. 2, 4, the VL structural domain of sequence any in 6,11,12,13,14,15,16,17 or 18.
3. conjugate according to claim 2, wherein the VH structural domain and the VL structural domain have following pairs of Sequence:
Pairs of SEQ ID NO.1 and SEQ ID NO.4 pairs of SEQ ID NO.3 and SEQ ID with SEQ ID NO.2 NO.6 pairs of SEQ ID NO.5 and SEQ ID NO.11 pairs of SEQ ID NO.7 and SEQ ID NO.12 pairs of SEQ ID NO.7 and SEQ ID NO.13 pairs of SEQ ID NO.7 and SEQ ID NO.14 pairs of SEQ ID NO.7 and SEQ It is ID NO.15 pairs of SEQ ID NO.7, SEQ ID NO.7 pairs of with SEQ ID NO.16, pairs of with SEQ ID NO.17 SEQ ID NO.7, with SEQ ID NO.18 pairs of SEQ ID NO.7, with SEQ ID NO.11 pairs of SEQ ID NO.8 and SEQ ID NO.12 pairs of SEQ ID NO.8 and SEQ ID NO.13 pairs of SEQ ID NO.8 and SEQ ID NO.14 pairs of SEQ ID NO.8, with SEQ ID NO.15 pairs of SEQ ID NO.8, it is pairs of with SEQ ID NO.16 SEQ ID NO.8 and SEQ ID NO.17 pairs of SEQ ID NO.8 and SEQ ID NO.18 pairs of SEQ ID NO.8, Pairs of SEQ ID NO.9 and SEQ ID NO.12 pairs of SEQ ID NO.9 and SEQ ID with SEQ ID NO.11 NO.13 pairs of SEQ ID NO.9, with SEQ ID NO.14 pairs of SEQ ID NO.9, it is pairs of with SEQ ID NO.15 SEQ ID NO.9 and SEQ ID NO.16 pairs of SEQ ID NO.9 and SEQ ID NO.17 pairs of SEQ ID NO.9, Pairs of SEQ ID NO.9 and SEQ ID NO.11 pairs of SEQ ID NO.10 and SEQ ID with SEQ ID NO.18 NO.12 pairs of SEQ ID NO.10, with SEQ ID NO.13 pairs of SEQ ID NO.10, it is pairs of with SEQ ID NO.14 SEQ ID NO.10 and SEQ ID NO.15 pairs of SEQ ID NO.10 and SEQ ID NO.16 pairs of SEQ ID The NO.10 and SEQ ID NO.17 pairs of SEQ ID NO.10 or SEQ ID NO.10 pairs of with SEQ ID NO.18.
4. conjugate according to any one of claim 1 to 3, wherein the antibody is complete antibody.
5. conjugate according to claim 1, wherein the antibody is humanization, removes immune or resurfacing.
6. conjugate according to claim 5, wherein the antibody is humanization below, removes immune or resurfacing Version: (i) include the VH structural domain SEQ ID NO.1 pairs of with VL structural domain SEQ ID NO.2 antibody；(ii)WO02/ J519 antibody disclosed in 098897；(iii) antibody secreted by hybridoma ATCC accession number HB-12109；(iv) include and VL The antibody of structural domain SEQ ID NO. 6 pairs of VH structural domain SEQ ID NO. 5；(v) J415 disclosed in WO02/098897 Antibody；(vi) antibody secreted by hybridoma ATCC accession number HB-12126.
7. conjugate according to claim 1, wherein the drugloading rate (p) is 1,2,3 or 4.
8. conjugate according to claim 7, the mixture including the antibody-drug conjugates compound, wherein institute Drug loading/the antibody stated in the mixture of antibody-drug conjugates compound is 2 to 5.
9. conjugate according to any one of claim 1 to 8 answering in the drug that preparation is used to use in the treatment With.
10. a kind of pharmaceutical composition includes conjugate described in any item of the claim 1 to 8 and pharmaceutically acceptable load Body or excipient.
11. pharmaceutical composition described in any one of claim 10 further includes the chemotherapeutics of therapeutically effective amount.
12. conjugate according to any one of claim 1 to 8 is in preparation for the proliferative disease in subject Purposes in drug used in treatment.
13. purposes described in claim 12, wherein the disease is cancer.
14. pharmaceutical composition described in any one of claim 10 is in preparation for treating the purposes in patient in the drug of cancer.
CN201380065405.7A 2012-10-12 2013-10-11 Tall and erect-anti-PSMA antibody conjugates of Pyrrolobenzodiazepines Expired - Fee Related CN105102003B (en)

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US201261712928P 2012-10-12 2012-10-12
US201261712924P 2012-10-12 2012-10-12
US61/712,928 2012-10-12
US61/712,924 2012-10-12
US201361784460P 2013-03-14 2013-03-14
US201361784525P 2013-03-14 2013-03-14
US201361784477P 2013-03-14 2013-03-14
US61/784,525 2013-03-14
US61/784,460 2013-03-14
US61/784,477 2013-03-14
PCT/EP2013/071341 WO2014057113A1 (en) 2012-10-12 2013-10-11 Pyrrolobenzodiazepine - anti-psma antibody conjugates

Publications (2)

Publication Number Publication Date
CN105102003A CN105102003A (en) 2015-11-25
CN105102003B true CN105102003B (en) 2019-03-05

Family

ID=49641717

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201380065405.7A Expired - Fee Related CN105102003B (en) 2012-10-12 2013-10-11 Tall and erect-anti-PSMA antibody conjugates of Pyrrolobenzodiazepines

Country Status (22)

Country Link
US (1) US10751346B2 (en)
EP (1) EP2906253B9 (en)
JP (1) JP6270859B2 (en)
KR (1) KR101986404B1 (en)
CN (1) CN105102003B (en)
AU (1) AU2013328619B2 (en)
BR (1) BR112015008173A2 (en)
CA (1) CA2887894C (en)
CY (1) CY1120916T1 (en)
DK (1) DK2906253T3 (en)
ES (1) ES2690067T3 (en)
HR (1) HRP20181646T2 (en)
HU (1) HUE041274T2 (en)
LT (1) LT2906253T (en)
MX (1) MX364326B (en)
NZ (1) NZ707486A (en)
PL (1) PL2906253T3 (en)
PT (1) PT2906253T (en)
RS (1) RS57694B1 (en)
SI (1) SI2906253T1 (en)
TR (1) TR201815418T4 (en)
WO (1) WO2014057113A1 (en)

Families Citing this family (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0819095D0 (en) 2008-10-17 2008-11-26 Spirogen Ltd Pyrrolobenzodiazepines
CN103068405A (en) 2010-04-15 2013-04-24 西雅图基因公司 Targeted pyrrolobenzodiazapine conjugates
CN102971329B (en) 2010-04-15 2016-06-29 麦迪穆有限责任公司 Pyrrolobenzodiazepines for treating proliferative disease is tall and erect
AU2012311505B2 (en) 2011-09-20 2016-09-29 Medimmune Limited Pyrrolobenzodiazepines as unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates
MX341523B (en) 2011-10-14 2016-08-24 Medimmune Ltd Pyrrolobenzodiazepines.
CN110183470A (en) 2011-10-14 2019-08-30 麦迪穆有限责任公司 Pyrrolobenzodiazepines are tall and erect
KR101961976B1 (en) 2011-10-14 2019-03-25 시애틀 지네틱스, 인크. Pyrrolobenzodiazepines and targeted conjugates
CA2850373C (en) 2011-10-14 2019-07-16 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
US9745303B2 (en) 2012-10-12 2017-08-29 Medimmune Limited Synthesis and intermediates of pyrrolobenzodiazepine derivatives for conjugation
LT2839860T (en) 2012-10-12 2019-07-10 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
AU2013328674B2 (en) 2012-10-12 2017-06-22 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
TR201815418T4 (en) 2012-10-12 2018-11-21 Adc Therapeutics Sa Pyrrolobenzodiazepine -anti-psma antibody conjugates.
US9567340B2 (en) 2012-12-21 2017-02-14 Medimmune Limited Unsymmetrical pyrrolobenzodiazepines-dimers for use in the treatment of proliferative and autoimmune diseases
EP2935268B2 (en) 2012-12-21 2021-02-17 MedImmune Limited Pyrrolobenzodiazepines and conjugates thereof
KR102066318B1 (en) 2013-03-13 2020-01-14 메디뮨 리미티드 Pyrrolobenzodiazepines and conjugates thereof
US9821074B2 (en) 2013-03-13 2017-11-21 Genentech, Inc. Pyrrolobenzodiazepines and conjugates thereof
GB201317982D0 (en) 2013-10-11 2013-11-27 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
GB201317981D0 (en) 2013-10-11 2013-11-27 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2015052532A1 (en) 2013-10-11 2015-04-16 Spirogen Sàrl Pyrrolobenzodiazepine-antibody conjugates
GB201416112D0 (en) * 2014-09-12 2014-10-29 Medimmune Ltd Pyrrolobenzodiazepines and conjugates thereof
ES2830385T3 (en) 2014-09-12 2021-06-03 Genentech Inc Anti-HER2 antibodies and immunoconjugates
AU2015352545B2 (en) 2014-11-25 2020-10-15 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
GB201506411D0 (en) 2015-04-15 2015-05-27 Bergenbio As Humanized anti-axl antibodies
GB201506393D0 (en) * 2015-04-15 2015-05-27 Berkel Patricius H C Van And Howard Philip W Site-specific antibody-drug conjugates
MA43345A (en) 2015-10-02 2018-08-08 Hoffmann La Roche PYRROLOBENZODIAZEPINE ANTIBODY-DRUG CONJUGATES AND METHODS OF USE
GB201601431D0 (en) * 2016-01-26 2016-03-09 Medimmune Ltd Pyrrolobenzodiazepines
GB201602356D0 (en) 2016-02-10 2016-03-23 Medimmune Ltd Pyrrolobenzodiazepine Conjugates
GB201602359D0 (en) 2016-02-10 2016-03-23 Medimmune Ltd Pyrrolobenzodiazepine Conjugates
GB201607478D0 (en) 2016-04-29 2016-06-15 Medimmune Ltd Pyrrolobenzodiazepine Conjugates
EP3496763A1 (en) * 2016-08-11 2019-06-19 Genentech, Inc. Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof
GB201617466D0 (en) 2016-10-14 2016-11-30 Medimmune Ltd Pyrrolobenzodiazepine conjugates
WO2018107109A1 (en) * 2016-12-09 2018-06-14 Abbvie Stemcentrx Llc Novel anti-kremen2 antibodies and methods of use
US11517627B2 (en) 2017-01-20 2022-12-06 Juno Therapeutics Gmbh Cell surface conjugates and related cell compositions and methods
GB201702031D0 (en) 2017-02-08 2017-03-22 Medlmmune Ltd Pyrrolobenzodiazepine-antibody conjugates
LT3544636T (en) 2017-02-08 2021-06-25 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
JP6704532B1 (en) 2017-02-08 2020-06-03 アーデーセー セラピューティクス ソシエテ アノニム Pyrrolobenzodiazepine antibody complex
US11654197B2 (en) 2017-03-29 2023-05-23 Legochem Biosciences, Inc. Pyrrolobenzodiazepine dimer prodrug and ligand-linker conjugate compound of the same
JP7284707B2 (en) 2017-04-07 2023-05-31 ジュノー セラピューティクス インコーポレイテッド Engineered Cells Expressing Prostate-Specific Membrane Antigen (PSMA) or Modified Forms Thereof and Related Methods
EP3612537B1 (en) 2017-04-18 2022-07-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2018193104A1 (en) 2017-04-20 2018-10-25 Adc Therapeutics Sa Combination therapy with an anti-cd25 antibody-drug conjugate
US20200129638A1 (en) * 2017-04-20 2020-04-30 Adc Therapeutics Sa Combination therapy with an anti-psma antibody-drug conjugate
WO2018193102A1 (en) 2017-04-20 2018-10-25 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
EP3638372A1 (en) 2017-06-14 2020-04-22 ADC Therapeutics SA Dosage regimes for the administration of an anti-cd25 adc
MX2020001880A (en) 2017-08-18 2021-07-06 Medimmune Ltd Pyrrolobenzodiazepine conjugates.
CN111164208B (en) 2017-09-29 2023-08-04 第一三共株式会社 Antibody-pyrrolobenzodiazepine derivative conjugates
GB201803342D0 (en) 2018-03-01 2018-04-18 Medimmune Ltd Methods
GB201806022D0 (en) 2018-04-12 2018-05-30 Medimmune Ltd Pyrrolobenzodiazepines and conjugates thereof
US20220347309A1 (en) 2018-12-19 2022-11-03 Adc Therapeutics Sa Pyrrolobenzodiazepine resistance
GB201820725D0 (en) 2018-12-19 2019-01-30 Adc Therapeutics Sarl Pyrrolobenzodiazepine resistance
JP2022516911A (en) 2019-01-03 2022-03-03 レゴケム バイオサイエンシズ， インク． Pyrrolobenzodiazepine dimer compounds with improved stability and their uses
KR20200084802A (en) * 2019-01-03 2020-07-13 주식회사 레고켐 바이오사이언스 Pyrrolobenzodiazepine dimer compounds with improved safety and its use
GB201908128D0 (en) 2019-06-07 2019-07-24 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
WO2021030358A1 (en) 2019-08-12 2021-02-18 Regeneron Pharmaceuticals, Inc. Macrophage stimulating 1 receptor (mst1r) variants and uses thereof
US20230201366A1 (en) * 2021-11-19 2023-06-29 Adc Therapeutics Sa Anti-psma conjugates
WO2023173096A1 (en) * 2022-03-11 2023-09-14 Sigma-Aldrich Co. Llc Pyrrolobenzodiazepine intermediates and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7514078B2 (en) * 2001-06-01 2009-04-07 Cornell Research Foundation, Inc. Methods of treating prostate cancer with anti-prostate specific membrane antigen antibodies

Family Cites Families (352)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3361742A (en) 1964-12-07 1968-01-02 Hoffmann La Roche 5-oxo-1h-pyrrolo-[2, 1-c][1, 4]-benzodiazepin-2-crylamides
US3523941A (en) 1967-03-06 1970-08-11 Hoffmann La Roche Benzodiazepine compounds and process for their preparation
US3524849A (en) 1967-10-27 1970-08-18 Hoffmann La Roche Process for the preparation of pyrrolo-benzodiazepine acrylamides and intermediates useful therein
JPS4843755B1 (en) 1969-06-26 1973-12-20
DE1965304A1 (en) 1968-12-30 1970-07-23 Fujisawa Pharmaceutical Co Benzdiazepinone compounds and processes for their preparation
IL33558A (en) 1968-12-30 1973-10-25 Fujisawa Pharmaceutical Co Antibiotic pyrrolo-benzodiazepine compound,its derivatives and processes for their production
JPS6053033B2 (en) 1976-12-28 1985-11-22 財団法人微生物化学研究会 New anticancer antibiotic mazethramycin and its production method
JPS585916B2 (en) 1977-12-27 1983-02-02 株式会社ミドリ十字 New benzodiazepine compounds
JPS5615289A (en) 1979-07-17 1981-02-14 Green Cross Corp:The Novel benzodiazepinnbased compound 3
JPS57131791A (en) 1980-12-31 1982-08-14 Fujisawa Pharmaceut Co Ltd Benzodiazepine derivative and its preparation
CA1173441A (en) 1981-02-27 1984-08-28 Hoffmann-La Roche Limited Imidazodiazepines
CA1185602A (en) 1981-02-27 1985-04-16 Emilio Kyburz Imidazodiazepines
CA1184175A (en) 1981-02-27 1985-03-19 Walter Hunkeler Imidazodiazepines
JPS58180487A (en) 1982-04-16 1983-10-21 Kyowa Hakko Kogyo Co Ltd Antibiotic dc-81 and its preparation
US4427588A (en) 1982-11-08 1984-01-24 Bristol-Myers Company Process for conversion of oxotomaymycin to tomaymycin
US4427587A (en) 1982-11-10 1984-01-24 Bristol-Myers Company Total synthesis of antitumor antibiotics BBM-2040A and BBM-2040B
JPS59152329A (en) 1983-02-17 1984-08-31 Green Cross Corp:The Local disorder inhibitor
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
FR2586683B1 (en) 1985-08-29 1988-07-01 Centre Nat Rech Scient NOVEL NEOTHRAMYCIN DERIVATIVES, THEIR PREPARATION PROCESS AND THEIR APPLICATION AS MEDICAMENTS
US5583024A (en) 1985-12-02 1996-12-10 The Regents Of The University Of California Recombinant expression of Coleoptera luciferase
JP2660201B2 (en) 1988-08-05 1997-10-08 塩野義製薬株式会社 Novel pyrrolo [1,4] benzodiazepine derivatives and senile dementia drugs
CA2023779A1 (en) 1989-08-23 1991-02-24 Margaret D. Moore Compositions and methods for detection and treatment of epstein-barr virus infection and immune disorders
JPH053790A (en) 1990-04-19 1993-01-14 Fujisawa Pharmaceut Co Ltd Dehydropeptidase-i
US5256643A (en) 1990-05-29 1993-10-26 The Government Of The United States Human cripto protein
AU9016591A (en) 1990-10-25 1992-05-26 Tanox Biosystems, Inc. Glycoproteins associated with membrane-bound immunoglobulins as antibody targets on B cells
US5543503A (en) 1991-03-29 1996-08-06 Genentech Inc. Antibodies to human IL-8 type A receptor
US5440021A (en) 1991-03-29 1995-08-08 Chuntharapai; Anan Antibodies to human IL-8 type B receptor
JP4156662B2 (en) 1991-03-29 2008-09-24 ジェネンテック・インコーポレーテッド Human PF4A receptor and use thereof
FR2676230B1 (en) 1991-05-07 1993-08-27 Centre Nat Rech Scient NOVEL PYRROLO [1,4] -BENZODIAZEPINES DERIVATIVES, PROCESS FOR THEIR PREPARATION AND MEDICAMENTS CONTAINING THEM.
JP3050424B2 (en) 1991-07-12 2000-06-12 塩野義製薬株式会社 Human endothelin receptor
US5264557A (en) 1991-08-23 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Polypeptide of a human cripto-related gene, CR-3
US5362852A (en) 1991-09-27 1994-11-08 Pfizer Inc. Modified peptide derivatives conjugated at 2-hydroxyethylamine moieties
US6153408A (en) 1991-11-15 2000-11-28 Institut Pasteur And Institut National De La Sante Et De La Recherche Medicale Altered major histocompatibility complex (MHC) determinant and methods of using the determinant
US6011146A (en) 1991-11-15 2000-01-04 Institut Pasteur Altered major histocompatibility complex (MHC) determinant and methods of using the determinant
GB9205051D0 (en) 1992-03-09 1992-04-22 Cancer Res Campaign Tech Pyrrolobenzodiazepine derivatives,their preparation,and compositions containing them
FR2696176B1 (en) 1992-09-28 1994-11-10 Synthelabo Piperidine derivatives, their preparation and their therapeutic application.
IL107366A (en) 1992-10-23 2003-03-12 Chugai Pharmaceutical Co Ltd Genes coding for megakaryocyte potentiator
US5644033A (en) 1992-12-22 1997-07-01 Health Research, Inc. Monoclonal antibodies that define a unique antigen of human B cell antigen receptor complex and methods of using same for diagnosis and treatment
US5801005A (en) 1993-03-17 1998-09-01 University Of Washington Immune reactivity to HER-2/neu protein for diagnosis of malignancies in which the HER-2/neu oncogene is associated
US5869445A (en) 1993-03-17 1999-02-09 University Of Washington Methods for eliciting or enhancing reactivity to HER-2/neu protein
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
GB9316162D0 (en) 1993-08-04 1993-09-22 Zeneca Ltd Fungicides
US5773223A (en) 1993-09-02 1998-06-30 Chiron Corporation Endothelin B1, (ETB1) receptor polypeptide and its encoding nucleic acid methods, and uses thereof
EP0647450A1 (en) 1993-09-09 1995-04-12 BEHRINGWERKE Aktiengesellschaft Improved prodrugs for enzyme mediated activation
US5750370A (en) 1995-06-06 1998-05-12 Human Genome Sciences, Inc. Nucleic acid encoding human endothlein-bombesin receptor and method of producing the receptor
JPH08336393A (en) 1995-04-13 1996-12-24 Mitsubishi Chem Corp Production of optically active gamma-substituted-beta-hydroxybutyric ester
US5707829A (en) 1995-08-11 1998-01-13 Genetics Institute, Inc. DNA sequences and secreted proteins encoded thereby
US20020193567A1 (en) 1995-08-11 2002-12-19 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
JP3646191B2 (en) 1996-03-19 2005-05-11 大塚製薬株式会社 Human gene
US6218519B1 (en) 1996-04-12 2001-04-17 Pro-Neuron, Inc. Compounds and methods for the selective treatment of cancer and bacterial infections
BR9710968A (en) 1996-05-17 2001-09-04 Schering Corp Human B cell antigens; related reagents
AU739028B2 (en) 1996-09-27 2001-10-04 Bristol-Myers Squibb Company Hydrolyzable prodrugs for delivery of anticancer drugs to metastatic cells
US6759509B1 (en) 1996-11-05 2004-07-06 Bristol-Myers Squibb Company Branched peptide linkers
US5945511A (en) 1997-02-20 1999-08-31 Zymogenetics, Inc. Class II cytokine receptor
US20030185830A1 (en) 1997-02-25 2003-10-02 Corixa Corporation Compositions and methods for the therapy and diagnosis of prostate cancer
US7033827B2 (en) 1997-02-25 2006-04-25 Corixa Corporation Prostate-specific polynucleotide compositions
EP1514876B1 (en) 1997-03-10 2013-05-08 The Regents of The University of California Antibody against prostate stem cell antigen (PSCA)
US6261791B1 (en) 1997-03-10 2001-07-17 The Regents Of The University Of California Method for diagnosing cancer using specific PSCA antibodies
US6541212B2 (en) 1997-03-10 2003-04-01 The Regents Of The University Of California Methods for detecting prostate stem cell antigen protein
US6555339B1 (en) 1997-04-14 2003-04-29 Arena Pharmaceuticals, Inc. Non-endogenous, constitutively activated human protein-coupled receptors
US6319688B1 (en) 1997-04-28 2001-11-20 Smithkline Beecham Corporation Polynucleotide encoding human sodium dependent phosphate transporter (IPT-1)
WO1998051824A1 (en) 1997-05-15 1998-11-19 Abbott Laboratories Reagents and methods useful for detecting disease of the urinary tract
US6890749B2 (en) 1997-05-15 2005-05-10 Abbott Laboratories Reagents and methods useful for detecting diseases of the prostate
US6602677B1 (en) 1997-09-19 2003-08-05 Promega Corporation Thermostable luciferases and methods of production
US20030060612A1 (en) 1997-10-28 2003-03-27 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
US20020034749A1 (en) 1997-11-18 2002-03-21 Billing-Medel Patricia A. Reagents and methods useful for detecting diseases of the breast
US6110695A (en) 1997-12-02 2000-08-29 The Regents Of The University Of California Modulating the interaction of the chemokine, B Lymphocyte Hemoattractant, and its Receptor, BLR1
WO2004031238A2 (en) 2002-10-03 2004-04-15 Mcgill Univeristy Antibodies and cyclic peptides which bind cea (carcinoembryonic antigen) and their use as cancer therapeutics
WO1999046284A2 (en) 1998-03-13 1999-09-16 The Burnham Institute Molecules that home to various selected organs or tissues
JP2002520000A (en) 1998-05-13 2002-07-09 エピミューン， インコーポレイテッド Expression vector and method of using the vector to stimulate an immune response
JP4560210B2 (en) 1998-05-22 2010-10-13 第一三共株式会社 Drug complex
US20020187472A1 (en) 2001-03-09 2002-12-12 Preeti Lal Steap-related protein
US20030064397A1 (en) 1998-05-22 2003-04-03 Incyte Genomics, Inc. Transmembrane protein differentially expressed in prostate and lung tumors
WO2002016429A2 (en) 2000-08-24 2002-02-28 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
EP1754995B1 (en) 1998-07-08 2012-04-04 E Ink Corporation Methods for achieving improved color in microencapsulted electrophoretic devices
WO2000012130A1 (en) 1998-08-27 2000-03-09 Smithkline Beecham Corporation Rp105 agonists and antagonists
DE69930328T2 (en) 1998-08-27 2006-12-14 Spirogen Ltd., Ryde Dimer pyrrolobenzodiazepines
GB9818731D0 (en) 1998-08-27 1998-10-21 Univ Portsmouth Compounds
GB9818732D0 (en) 1998-08-27 1998-10-21 Univ Portsmouth Collection of compounds
GB9818730D0 (en) 1998-08-27 1998-10-21 Univ Portsmouth Collections of compounds
JP4689781B2 (en) 1998-09-03 2011-05-25 独立行政法人科学技術振興機構 Amino acid transport protein and its gene
AU5963699A (en) 1998-10-02 2000-04-26 Mcmaster University Spliced form of (erb)b-2/neu oncogene
US20030091580A1 (en) 2001-06-18 2003-05-15 Mitcham Jennifer L. Compositions and methods for the therapy and diagnosis of ovarian cancer
US6962980B2 (en) 1999-09-24 2005-11-08 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US20020119158A1 (en) 1998-12-17 2002-08-29 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US6468546B1 (en) 1998-12-17 2002-10-22 Corixa Corporation Compositions and methods for therapy and diagnosis of ovarian cancer
US6858710B2 (en) 1998-12-17 2005-02-22 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
AU776672B2 (en) 1998-12-30 2004-09-16 Beth Israel Deaconess Medical Center Characterization of a calcium channel family
US20030190669A1 (en) 1998-12-30 2003-10-09 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
BR0007840A (en) 1999-01-29 2002-01-22 Corixa Corp Her-2 / neu fusion proteins
GB9905124D0 (en) 1999-03-05 1999-04-28 Smithkline Beecham Biolog Novel compounds
AU3395900A (en) 1999-03-12 2000-10-04 Human Genome Sciences, Inc. Human lung cancer associated gene sequences and polypeptides
US7312303B2 (en) 1999-05-11 2007-12-25 Genentech, Inc. Anti-PRO4980 antibodies
US6268488B1 (en) 1999-05-25 2001-07-31 Barbas, Iii Carlos F. Prodrug activation using catalytic antibodies
WO2000075655A1 (en) 1999-06-03 2000-12-14 Takeda Chemical Industries, Ltd. Screening method with the use of cd100
DK2283866T3 (en) 1999-06-25 2015-05-18 Genentech Inc METHODS OF TREATMENT USING ANTI-ERBB ANTIBODY-MAYTANSINOID CONJUGATES
US6302318B1 (en) 1999-06-29 2001-10-16 General Electric Company Method of providing wear-resistant coatings, and related articles
US20030119113A1 (en) 1999-07-20 2003-06-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7297770B2 (en) 1999-08-10 2007-11-20 Genentech, Inc. PRO6496 polypeptides
US7294696B2 (en) 1999-08-17 2007-11-13 Genentech Inc. PRO7168 polypeptides
US6909006B1 (en) 1999-08-27 2005-06-21 Spirogen Limited Cyclopropylindole derivatives
JP3951035B2 (en) 1999-09-01 2007-08-01 ジェネンテック・インコーポレーテッド Secreted and transmembrane polypeptides and nucleic acids encoding them
US20030206918A1 (en) 1999-09-10 2003-11-06 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US20030129192A1 (en) 1999-09-10 2003-07-10 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US20030232056A1 (en) 1999-09-10 2003-12-18 Corixa Corporation Compositions and methods for the therapy and diagnosis of ovarian cancer
US6750054B2 (en) 2000-05-18 2004-06-15 Lexicon Genetics Incorporated Human semaphorin homologs and polynucleotides encoding the same
IL149116A0 (en) 1999-10-29 2002-11-10 Genentech Inc Anti-prostate stem cell antigen (psca) antibody compositions and methods of use
ES2568625T3 (en) 1999-11-29 2016-05-03 The Trustees Of Columbia University In The City Of New York Isolation of five novel genes encoding new Fc receptor-type melanomas involved in the pathogenesis of lymphoma / melanoma
AU1807401A (en) 1999-11-30 2001-06-12 Corixa Corporation Compositions and methods for therapy and diagnosis of breast cancer
CA2393738A1 (en) 1999-12-10 2001-06-14 Epimmune Inc. Inducing cellular immune responses to her2/neu using peptide and nucleic acid compositions
EP1857466B1 (en) 1999-12-23 2010-10-20 ZymoGenetics, Inc. Soluble interleukin-20 receptor
US6610286B2 (en) 1999-12-23 2003-08-26 Zymogenetics, Inc. Method for treating inflammation using soluble receptors to interleukin-20
NZ502058A (en) 1999-12-23 2003-11-28 Ovita Ltd Isolated mutated nucleic acid molecule for regulation of ovulation rate
WO2001046261A1 (en) 1999-12-23 2001-06-28 Zymogenetics, Inc. Method for treating inflammation
US20040001827A1 (en) 2002-06-28 2004-01-01 Dennis Mark S. Serum albumin binding peptides for tumor targeting
CA2390691C (en) 1999-12-24 2016-05-10 Genentech, Inc. Methods and compositions for prolonging elimination half-times of bioactive compounds
US7294695B2 (en) 2000-01-20 2007-11-13 Genentech, Inc. PRO10268 polypeptides
US20030224379A1 (en) 2000-01-21 2003-12-04 Tang Y. Tom Novel nucleic acids and polypeptides
WO2001053463A2 (en) 2000-01-21 2001-07-26 Corixa Corporation COMPOUNDS AND METHODS FOR PREVENTION AND TREATMENT OF HER-2/neu ASSOCIATED MALIGNANCIES
AU2001243142A1 (en) 2000-02-03 2001-08-14 Hyseq, Inc. Novel nucleic acids and polypeptides
US20030104562A1 (en) 2000-02-11 2003-06-05 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20030219806A1 (en) 2000-02-22 2003-11-27 Millennium Pharmaceuticals, Inc. Novel 18607, 15603, 69318, 12303, 48000, 52920, 5433, 38554, 57301, 58324, 55063, 52991, 59914, 59921 and 33751 molecules and uses therefor
US20020142377A1 (en) 2000-02-22 2002-10-03 Glucksmann Maria Alexandra 18607, a novel human calcium channel
US20040052793A1 (en) 2001-02-22 2004-03-18 Carter Paul J. Caspase activivated prodrugs therapy
US20040002068A1 (en) 2000-03-01 2004-01-01 Corixa Corporation Compositions and methods for the detection, diagnosis and therapy of hematological malignancies
US20040005561A1 (en) 2000-03-01 2004-01-08 Corixa Corporation Compositions and methods for the detection, diagnosis and therapy of hematological malignancies
EP1261743A2 (en) 2000-03-07 2002-12-04 Hyseq, Inc. Novel nucleic acids and polypeptides
JP2004521602A (en) 2000-03-24 2004-07-22 ファハリ サッチオグリュ Novel prostate-specific or testis-specific nucleic acid molecules, polypeptides, and diagnostic and therapeutic methods
US20030186889A1 (en) 2000-03-31 2003-10-02 Wolf-Georg Forssmann Diagnostic and medicament for analysing the cell surface proteome of tumour and inflammatory cells and for treating tumorous and inflammatory diseases, preferably using a specific chemokine receptor analysis and the chemokine receptor-ligand interaction
WO2001075177A2 (en) 2000-04-03 2001-10-11 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Tumor markers in ovarian cancer
AU2001249885A1 (en) 2000-04-07 2001-10-23 Arena Pharmaceuticals, Inc. Non-endogenous, constitutively activated known g protein-coupled receptors
AU2001274888A1 (en) 2000-05-19 2001-12-03 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies
US20020051990A1 (en) 2000-06-09 2002-05-02 Eric Ople Novel gene targets and ligands that bind thereto for treatment and diagnosis of ovarian carcinomas
AU2001268471A1 (en) 2000-06-16 2002-01-02 Incyte Genomics, Inc. G-protein coupled receptors
JP2004528003A (en) 2000-06-30 2004-09-16 インサイト・ゲノミックス・インコーポレイテッド Extracellular matrix and cell adhesion molecules
WO2002002587A1 (en) 2000-06-30 2002-01-10 Human Genome Sciences, Inc. B7-like polynucleotides, polypeptides, and antibodies
MXPA02012749A (en) 2000-06-30 2003-10-06 Amgen Inc B7-like molecules and uses thereof.
WO2002006339A2 (en) 2000-07-03 2002-01-24 Curagen Corporation Proteins and nucleic acids encoding same
US20040044179A1 (en) 2000-07-25 2004-03-04 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US6891030B2 (en) 2000-07-27 2005-05-10 Mayo Foundation For Medical Education And Research T-cell immunoregulatory molecule
CA2417671A1 (en) 2000-07-28 2002-02-07 Ulrich Wissenbach Trp8, trp9 and trp10, novel markers for cancer
US7229623B1 (en) 2000-08-03 2007-06-12 Corixa Corporation Her-2/neu fusion proteins
HUP0600780A2 (en) 2000-08-14 2007-01-29 Corixa Corp Compositions and methods for the therapy and diagnosis of her-2/neu-associated malignancies
WO2002013847A2 (en) 2000-08-14 2002-02-21 Corixa Corporation Methods for diagnosis and therapy of hematological and virus-associated malignancies
GB0020953D0 (en) 2000-08-24 2000-10-11 Smithkline Beecham Biolog Vaccine
CA2421949A1 (en) 2000-09-11 2002-03-21 Hyseq, Inc. Novel nucleic acids and polypeptides
US7491797B2 (en) 2000-09-15 2009-02-17 Genentech, Inc. PRO6308 polypeptide
ATE460429T1 (en) 2000-09-15 2010-03-15 Zymogenetics Inc POLYPEPTIDES CONTAINING EXTRACELLULAR DOMAIN OF IL-20RA AND/OR IL-20RB
US6613567B1 (en) 2000-09-15 2003-09-02 Isis Pharmaceuticals, Inc. Antisense inhibition of Her-2 expression
AU2001292724B2 (en) 2000-09-18 2005-05-26 Biogen Idec Ma Inc. CRIPTO mutant and uses thereof
UA83458C2 (en) 2000-09-18 2008-07-25 Байоджен Айдек Ма Інк. The isolated polypeptide baff-r (the receptor of the factor of activation of b-cells of the family tnf)
DK1320522T3 (en) 2000-09-19 2006-04-03 Moses Lee Achiral analogs of CC-1065 and of duocarmycins as well as preparations and methods for their use
CA2425569A1 (en) 2000-10-13 2002-04-18 Eos Biotechnology, Inc. Methods of diagnosis of prostate cancer, compositions and methods of screening for modulators of prostate cancer
CA2428242A1 (en) 2000-11-07 2002-05-16 Zymogenetics, Inc. Human tumor necrosis factor receptor
US20020150573A1 (en) 2000-11-10 2002-10-17 The Rockefeller University Anti-Igalpha-Igbeta antibody for lymphoma therapy
US20040018194A1 (en) 2000-11-28 2004-01-29 Francisco Joseph A. Recombinant anti-CD30 antibodies and uses thereof
WO2002061087A2 (en) 2000-12-19 2002-08-08 Lifespan Biosciences, Inc. Antigenic peptides, such as for g protein-coupled receptors (gpcrs), antibodies thereto, and systems for identifying such antigenic peptides
AU2002243495A1 (en) 2001-01-12 2002-07-24 University Of Medicine And Dentistry Of New Jersey Bone morphogenetic protein-2 in the treatment and diagnosis of cancer
US20030119125A1 (en) 2001-01-16 2003-06-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20030119126A1 (en) 2001-01-16 2003-06-26 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7754208B2 (en) 2001-01-17 2010-07-13 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
MXPA03006617A (en) 2001-01-24 2004-12-02 Protein Design Labs Inc Methods of diagnosis of breast cancer, compositions and methods of screening for modulators of breast cancer.
US20030073144A1 (en) 2001-01-30 2003-04-17 Corixa Corporation Compositions and methods for the therapy and diagnosis of pancreatic cancer
US20040170994A1 (en) 2001-02-12 2004-09-02 Callen David Frederick DNA sequences for human tumour suppressor genes
WO2002071928A2 (en) 2001-03-14 2002-09-19 Millennium Pharmaceuticals, Inc. Nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer
EP1243276A1 (en) 2001-03-23 2002-09-25 Franciscus Marinus Hendrikus De Groot Elongated and multiple spacers containing activatible prodrugs
AU2002311787A1 (en) 2001-03-28 2002-10-15 Zycos Inc. Translational profiling
US6362331B1 (en) 2001-03-30 2002-03-26 Council Of Scientific And Industrial Research Process for the preparation of antitumor agents
WO2003008537A2 (en) 2001-04-06 2003-01-30 Mannkind Corporation Epitope sequences
US6820011B2 (en) 2001-04-11 2004-11-16 The Regents Of The University Of Colorado Three-dimensional structure of complement receptor type 2 and uses thereof
MXPA03009510A (en) 2001-04-17 2005-04-29 Univ Arkansas Repeat sequences of the ca125 gene and their use for diagnostic and therapeutic interventions.
CA2444691A1 (en) 2001-04-18 2002-10-31 Protein Design Labs, Inc. Methods of diagnosis of lung cancer, compositions and methods of screening for modulators of lung cancer
DE60230868D1 (en) 2001-04-26 2009-03-05 Biogen Inc CRIPTOBLOCKING ANTIBODIES AND THEIR USE
AU2002334799B2 (en) 2001-04-26 2009-05-07 Biogen Ma Inc. Cripto-specific antibodies
US6884869B2 (en) 2001-04-30 2005-04-26 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
WO2002089747A2 (en) 2001-05-09 2002-11-14 Corixa Corporation Compositions and methods for the therapy and diagnosis of prostate cancer
AU2002344326A1 (en) 2001-05-11 2002-11-25 Sloan-Kettering Institute For Cancer Research Nucleic acid sequence encoding ovarian antigen, ca125, and uses thereof
IL158920A0 (en) 2001-05-24 2004-05-12 Zymogenetics Inc Taci-immunoglobulin fusion proteins
US7157558B2 (en) 2001-06-01 2007-01-02 Genentech, Inc. Polypeptide encoded by a polynucleotide overexpresses in tumors
US7045605B2 (en) 2001-06-01 2006-05-16 Cornell Research Foundation, Inc. Modified antibodies to prostate-specific membrane antigen and uses thereof
WO2002098358A2 (en) 2001-06-04 2002-12-12 Eos Biotechnology, Inc. Methods of diagnosis and treatment of androgen-dependent prostate cancer, prostate cancer undergoing androgen-withdrawal, and androgen-independent prostate cancer
WO2003000842A2 (en) 2001-06-04 2003-01-03 Curagen Corporation Novel proteins and nucleic acids encoding same
US20050112568A1 (en) 2001-06-05 2005-05-26 Lori Friedman Dgks as modifiers of the p53 pathwha and methods of use
ATE483976T1 (en) 2001-06-05 2010-10-15 Exelixis Inc GFATS AS P53 PATHWAY MODIFIERS AND METHODS OF USE
US7235358B2 (en) 2001-06-08 2007-06-26 Expression Diagnostics, Inc. Methods and compositions for diagnosing and monitoring transplant rejection
US7125663B2 (en) 2001-06-13 2006-10-24 Millenium Pharmaceuticals, Inc. Genes, compositions, kits and methods for identification, assessment, prevention, and therapy of cervical cancer
US7189507B2 (en) 2001-06-18 2007-03-13 Pdl Biopharma, Inc. Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer
MXPA03011979A (en) 2001-06-18 2005-04-08 Eos Biotechnology Inc Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer.
US7705120B2 (en) 2001-06-21 2010-04-27 Millennium Pharmaceuticals, Inc. Compositions, kits, and methods for identification, assessment, prevention, and therapy of breast cancer
WO2003002717A2 (en) 2001-06-28 2003-01-09 Schering Corporation Biological activity of ak155
WO2003004529A2 (en) 2001-07-02 2003-01-16 Licentia Ltd. Ephrin-tie receptor materials and methods
US20040076955A1 (en) 2001-07-03 2004-04-22 Eos Biotechnology, Inc. Methods of diagnosis of bladder cancer, compositions and methods of screening for modulators of bladder cancer
WO2003003984A2 (en) 2001-07-05 2003-01-16 Curagen Corporation Novel proteins and nucleic acids encoding same
US7446185B2 (en) 2001-07-18 2008-11-04 The Regents Of The University Of California Her2/neu target antigen and use of same to stimulate an immune response
US20030108963A1 (en) 2001-07-25 2003-06-12 Millennium Pharmaceuticals, Inc. Novel genes, compositions, kit, and methods for identification, assessment, prevention and therapy of prostate cancer
MXPA04001050A (en) 2001-08-03 2004-07-08 Genentech Inc Tacis and br3 polypeptides and uses thereof.
AU2002324700A1 (en) 2001-08-14 2003-03-03 Bayer Ag Nucleic acid and amino acid sequences involved in pain
US20030092013A1 (en) 2001-08-16 2003-05-15 Vitivity, Inc. Diagnosis and treatment of vascular disease
WO2003018621A2 (en) 2001-08-23 2003-03-06 Oxford Biomedica (Uk) Limited Genes
AU2002357643A1 (en) 2001-08-29 2003-04-14 Vanderbilt University The human mob-5 (il-24) receptors and uses thereof
US20030124579A1 (en) 2001-09-05 2003-07-03 Eos Biotechnology, Inc. Methods of diagnosis of ovarian cancer, compositions and methods of screening for modulators of ovarian cancer
ES2537074T3 (en) 2001-09-06 2015-06-02 Agensys, Inc. Nucleic acid and corresponding protein called STEAP-1 useful in the treatment and detection of cancer
AU2002330039A1 (en) 2001-09-17 2003-04-01 Eos Biotechnology, Inc. Methods of diagnosis of cancer compositions and methods of screening for modulators of cancer
US20050004017A1 (en) 2001-09-18 2005-01-06 Yuval Reiss Methods and compositions for treating hcap associated diseases
DE60238143D1 (en) 2001-09-18 2010-12-09 Genentech Inc COMPOSITIONS AND METHODS FOR THE DIAGNOSIS OF TUMORS
WO2003025148A2 (en) 2001-09-19 2003-03-27 Nuvelo, Inc. Novel nucleic acids and polypeptides
WO2003026577A2 (en) 2001-09-24 2003-04-03 Seattle Genetics, Inc. P-amidobenzylethers in drug delivery agents
US7091186B2 (en) 2001-09-24 2006-08-15 Seattle Genetics, Inc. p-Amidobenzylethers in drug delivery agents
US20030077644A1 (en) 2001-09-28 2003-04-24 Bing Yang Diagnosis and treatment of diseases caused by mutations in CD72
WO2003029421A2 (en) 2001-10-03 2003-04-10 Origene Technologies, Inc. Regulated breast cancer genes
US20050130117A1 (en) 2001-10-03 2005-06-16 Davis Cong L. Modulators of lymphocyte activation and migration
US20050089957A1 (en) 2001-10-19 2005-04-28 Audrey Goddard Compositions and methods for the diagnosis and treatment of inflammatory bowel disorders
US20050123925A1 (en) 2002-11-15 2005-06-09 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
WO2003035846A2 (en) 2001-10-24 2003-05-01 National Jewish Medical And Research Center Structure of tall-1 and its cognate receptor
US7405192B2 (en) 2001-10-31 2008-07-29 Alcon, Inc. Bone morphogenic proteins (BMP), BMP receptors and BMP binding proteins and their use in the diagnosis and treatment of glaucoma
US20030232350A1 (en) 2001-11-13 2003-12-18 Eos Biotechnology, Inc. Methods of diagnosis of cancer, compositions and methods of screening for modulators of cancer
WO2003042661A2 (en) 2001-11-13 2003-05-22 Protein Design Labs, Inc. Methods of diagnosis of cancer, compositions and methods of screening for modulators of cancer
JP2005511627A (en) 2001-11-20 2005-04-28 シアトル ジェネティクス，インコーポレーテッド Treatment of immunological diseases using anti-CD30 antibodies
US7344843B2 (en) 2001-11-29 2008-03-18 Serono Genetics Institute S.A. Agonists and antagonists of prolixin for the treatment of metabolic disorders
AU2002349784A1 (en) 2001-12-03 2003-06-17 Asahi Kasei Pharma Corporation Nf-kappab activating genes
EP1504099A4 (en) 2001-12-10 2006-05-10 Nuvelo Inc Novel nucleic acids and polypeptides
US20030134790A1 (en) 2002-01-11 2003-07-17 University Of Medicine And Dentistry Of New Jersey Bone Morphogenetic Protein-2 And Bone Morphogenetic Protein-4 In The Treatment And Diagnosis Of Cancer
CA2473549C (en) 2002-01-16 2011-02-15 Japan Science And Technology Agency Moving-image holographic reproducing device and color moving-image holographic reproducing device
US7452675B2 (en) 2002-01-25 2008-11-18 The Queen's Medical Center Methods of screening for TRPM4b modulators
JP2005526501A (en) 2002-02-21 2005-09-08 デューク・ユニヴァーシティ Reagents and therapeutic methods for autoimmune diseases
JP2005535290A (en) 2002-02-22 2005-11-24 ジェネンテック・インコーポレーテッド Compositions and methods for the treatment of immune related diseases
WO2003074662A2 (en) 2002-03-01 2003-09-12 Exelixis, Inc. SCDs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE
WO2003104399A2 (en) 2002-06-07 2003-12-18 Avalon Pharmaceuticals, Inc Cancer-linked gene as target for chemotherapy
WO2003101400A2 (en) 2002-06-04 2003-12-11 Avalon Pharmaceuticals, Inc. Cancer-linked gene as target for chemotherapy
US6660856B2 (en) 2002-03-08 2003-12-09 Kaohsiung Medical University Synthesis of pyrrolo[2,1-c][1,4]benzodiazepine analogues
EP2258712A3 (en) 2002-03-15 2011-05-04 Multicell Immunotherapeutics, Inc. Compositions and Methods to Initiate or Enhance Antibody and Major-histocompatibility Class I or Class II-restricted T Cell Responses by Using Immunomodulatory, Non-coding RNA Motifs
WO2004000997A2 (en) 2002-03-19 2003-12-31 Curagen Corporation Therapeutic polypeptides, nucleic acids encoding same, and methods of use
WO2003081210A2 (en) 2002-03-21 2003-10-02 Sunesis Pharmaceuticals, Inc. Identification of kinase inhibitors
US7193069B2 (en) 2002-03-22 2007-03-20 Research Association For Biotechnology Full-length cDNA
US7317087B2 (en) 2002-03-25 2008-01-08 The Uab Research Foundation Members of the FC receptor homolog gene family (FCRH1-3, 6), related reagents, and uses thereof
WO2003083074A2 (en) 2002-03-28 2003-10-09 Idec Pharmaceuticals Corporation Novel gene targets and ligands that bind thereto for treatment and diagnosis of colon carcinomas
US20030194704A1 (en) 2002-04-03 2003-10-16 Penn Sharron Gaynor Human genome-derived single exon nucleic acid probes useful for gene expression analysis two
JP2005534287A (en) 2002-04-05 2005-11-17 アジェンシス， インコーポレイテッド Nucleic acids named 98P4B6 and corresponding proteins useful in the treatment and detection of cancer
US20040101874A1 (en) 2002-04-12 2004-05-27 Mitokor Inc. Targets for therapeutic intervention identified in the mitochondrial proteome
EP2011886A3 (en) 2002-04-16 2009-02-11 Genentech, Inc. Compositions and methods for the diagnosis and treatment of tumor
AU2003239158A1 (en) 2002-04-17 2003-11-03 Baylor College Of Medicine Aib1 as a prognostic marker and predictor of resistance to encocrine therapy
WO2003093444A2 (en) 2002-05-03 2003-11-13 Incyte Corporation Transporters and ion channels
EP1572925A4 (en) 2002-05-15 2007-08-15 Avalon Pharmaceuticals Cancer-linked gene as target for chemotherapy
AU2003232453A1 (en) 2002-05-30 2003-12-19 David K. Bol Human solute carrier family 7 member 11 (hslc7a11)
AU2003240495A1 (en) 2002-06-04 2003-12-19 Incyte Corporation Diagnostics markers for lung cancer
AU2003242633A1 (en) 2002-06-06 2003-12-22 Molecular Engines Laboratories Dudulin genes, non-human animal model: uses in human hematological disease
JP4594085B2 (en) 2002-06-06 2010-12-08 オンコセラピー・サイエンス株式会社 Genes and polypeptides related to human colon cancer
AU2003245441A1 (en) 2002-06-12 2003-12-31 Avalon Pharmaceuticals, Inc. Cancer-linked gene as target for chemotherapy
AU2003247576A1 (en) 2002-06-18 2003-12-31 Archemix Corp. Aptamer-toxin molecules and methods for using same
US20040249130A1 (en) 2002-06-18 2004-12-09 Martin Stanton Aptamer-toxin molecules and methods for using same
EP1539218A4 (en) 2002-06-20 2007-08-22 Univ California Compositions and methods for modulating lymphocyte activity
EP2365004B1 (en) 2002-06-21 2016-01-06 Johns Hopkins University School of Medicine Membrane associated tumor endothelium markers
DE10229834A1 (en) 2002-07-03 2004-01-29 Zinser Textilmaschinen Gmbh Drafting system for spinning machines with a downstream compacting device
AU2003281515A1 (en) 2002-07-19 2004-02-09 Cellzome Ag Protein complexes of cellular networks underlying the development of cancer and other diseases
CA2492447A1 (en) 2002-07-25 2004-02-05 Genentech, Inc. Taci antibodies and uses thereof
ES2544527T3 (en) 2002-07-31 2015-09-01 Seattle Genetics, Inc. Drug conjugates and their use to treat cancer, an autoimmune disease or an infectious disease
US20050180972A1 (en) 2002-07-31 2005-08-18 Wahl Alan F. Anti-CD20 antibody-drug conjugates for the treatment of cancer and immune disorders
JP2004121218A (en) 2002-08-06 2004-04-22 Jenokkusu Soyaku Kenkyusho:Kk Method for testing bronchial asthma or chronic obstructive pulmonary disease
AU2003251471A1 (en) 2002-08-06 2004-02-25 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with human cxc chemokine receptor 5(cxcr5)
EP1578371A4 (en) 2002-08-19 2009-05-20 Genentech Inc Compositions and methods for the diagnosis and treatment of tumor
WO2004020583A2 (en) 2002-08-27 2004-03-11 Bristol-Myers Squibb Company Polynucleotide predictor set for identifying protein tyrosine kinase modulators
WO2004020595A2 (en) 2002-08-29 2004-03-11 Five Prime Therapeutics, Inc. Novel human polypeptides encoded by polynucleotides
WO2004019993A1 (en) 2002-08-30 2004-03-11 Ramot At Tel Aviv University Ltd. Self-immolative dendrimers releasing many active moieties upon a single activating event
AU2002951346A0 (en) 2002-09-05 2002-09-26 Garvan Institute Of Medical Research Diagnosis of ovarian cancer
CN1691964A (en) 2002-09-06 2005-11-02 曼康公司 Epitope sequences
US20060134109A1 (en) 2002-09-09 2006-06-22 Nura Inc. G protein coupled receptors and uses thereof
JP2004113151A (en) 2002-09-27 2004-04-15 Sankyo Co Ltd Oncogene and its application
CA2501131A1 (en) 2002-10-04 2004-04-22 Van Andel Research Institute Molecular sub-classification of kidney tumors and the discovery of new diagnostic markers
US20040138269A1 (en) 2002-10-11 2004-07-15 Sugen, Inc. Substituted pyrroles as kinase inhibitors
AU2003295401B2 (en) 2002-11-08 2010-04-29 Genentech, Inc. Compositions and methods for the treatment of natural killer cell related diseases
WO2004044178A2 (en) 2002-11-13 2004-05-27 Genentech, Inc. Methods and compositions for diagnosing dysplasia
EP1560599A1 (en) 2002-11-14 2005-08-10 Syntarga B.V. Prodrugs built as multiple self-elimination-release spacers
GB0226593D0 (en) 2002-11-14 2002-12-24 Consultants Ltd Compounds
AU2003298650B2 (en) 2002-11-15 2010-03-11 Musc Foundation For Research Development Complement receptor 2 targeted complement modulators
WO2004045553A2 (en) 2002-11-15 2004-06-03 The Board Of Trustees Of The University Of Arkansas Ca125 gene and its use for diagnostic and therapeutic interventions
US20080213166A1 (en) 2002-11-20 2008-09-04 Biogen Idec Inc. Novel Gene Targets and Ligands that Bind Thereto for Treatment and Diagnosis of Colon Carcinomas
WO2004047749A2 (en) 2002-11-21 2004-06-10 University Of Utah Research Foundation Purinergic modulation of smell
US20040253606A1 (en) 2002-11-26 2004-12-16 Protein Design Labs, Inc. Methods of detecting soft tissue sarcoma, compositions and methods of screening for soft tissue sarcoma modulators
EP1581641A4 (en) 2002-12-06 2006-11-15 Diadexus Inc Compositions, splice variants and methods relating to ovarian specific genes and proteins
US20040157278A1 (en) 2002-12-13 2004-08-12 Bayer Corporation Detection methods using TIMP 1
US7276372B2 (en) 2002-12-20 2007-10-02 Pdl Biopharma, Inc. Antibodies against GPR64 and uses thereof
AU2003299819A1 (en) 2002-12-23 2004-07-22 Human Genome Sciences, Inc. Neutrokine-alpha conjugate, neutrokine-alpha complex, and uses thereof
EP1597558A2 (en) 2003-01-08 2005-11-23 Bristol-Myers Squibb Company Biomarkers and methods for determining sensitivity to epidermal growth factor receptor modulators
US20050227301A1 (en) 2003-01-10 2005-10-13 Polgen Cell cycle progression proteins
US20050181375A1 (en) 2003-01-10 2005-08-18 Natasha Aziz Novel methods of diagnosis of metastatic cancer, compositions and methods of screening for modulators of metastatic cancer
EP1583820A4 (en) 2003-01-14 2007-07-18 Bristol Myers Squibb Co Polynucleotides and polypeptides associated with the nf-kb pathway
JP2007520996A (en) 2003-01-15 2007-08-02 ミレニアム・ファーマシューティカルズ・インコーポレイテッド 44390, 54181, 211, 5687, 884, 1405, 636, 4421, 5410, 30905, 2045, 16405, 18560, 2047, 33751, 52721, 14063, 20739, 32544, 43239, 44373, 51164, 53010, 16852, 1587, 2207, 22245, 2387, 52908, 69112, 14990, 18547, 115, 579, 15985, 15625, 760, 18603, 2395, 2554, 8675, 32720, 4809, 14303, 16816, 17827, 32620, 577, 619, 1423, 2158, 8263, 15402, 16209, 16386, 21165, 30911, 41897, 1643, 2543, 9626, 13 31,32409,84260,2882,8203,32678 or methods and compositions for treating urological disorders using 55,053
JP2009520459A (en) 2003-02-14 2009-05-28 サイグレス ディスカバリー， インコーポレイテッド Therapeutic target in cancer
US20030224411A1 (en) 2003-03-13 2003-12-04 Stanton Lawrence W. Genes that are up- or down-regulated during differentiation of human embryonic stem cells
EP1608664B1 (en) 2003-03-31 2009-01-28 Council of Scientific and Industrial Research Non-cross-linking pyrrolo[2,1-c][1,4]benzodiazepines as potential antitumour agents and process thereof
GB0321295D0 (en) 2003-09-11 2003-10-15 Spirogen Ltd Synthesis of protected pyrrolobenzodiazepines
GB0416511D0 (en) 2003-10-22 2004-08-25 Spirogen Ltd Pyrrolobenzodiazepines
AU2004284075A1 (en) 2003-10-22 2005-05-06 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Pyrrolobenzodiazepine derivatives, compositions comprising the same and methods related thereto
SG149815A1 (en) 2003-11-06 2009-02-27 Seattle Genetics Inc Monomethylvaline compounds capable of conjugation to ligands
EP1720908A2 (en) 2004-02-17 2006-11-15 Absalus, Inc. Super-humanized antibodies against respiratory syncytial virus
EP1718667B1 (en) 2004-02-23 2013-01-09 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
GB0404577D0 (en) 2004-03-01 2004-04-07 Spirogen Ltd Pyrrolobenzodiazepines
GB0404574D0 (en) 2004-03-01 2004-04-07 Spirogen Ltd Amino acids
SI1720881T1 (en) 2004-03-01 2013-04-30 Spirogen Sarl 11-hydroxy-5h-pyrrolos2,1-ccs1,4cbenzodiazepin-5-one derivatives as key intermediates for the preparation of c2 substituted pyrrolobenzodiazepins
GB0404578D0 (en) 2004-03-01 2004-04-07 Spirogen Ltd Pyrrolobenzodiazepines
DE102004010943A1 (en) 2004-03-03 2005-09-29 Degussa Ag Process for the preparation of N-protected 4-ketproline derivatives
JP5166861B2 (en) 2004-03-09 2013-03-21 スピロゲン リミティッド Pyrrolobenzodiazepine
FR2869231B1 (en) 2004-04-27 2008-03-14 Sod Conseils Rech Applic THERAPEUTIC COMPOSITION CONTAINING AT LEAST ONE PYRROLOBENZODIAZEPINE DERIVATIVE AND FLUDARABINE
GB0410725D0 (en) 2004-05-13 2004-06-16 Spirogen Ltd Pyrrolobenzodiazepine therapeutic agents
CN101065151B (en) 2004-09-23 2014-12-10 健泰科生物技术公司 Cysteine engineered antibodies and conjugates
EP1831418A2 (en) 2004-12-24 2007-09-12 Showa Denko Kabushiki Kaisha Production method of thermoelectric semiconductor alloy, thermoelectric conversion module and thermoelectric power generating device
EP1844074B1 (en) 2005-02-03 2013-04-24 Antitope Limited Human antibodies and proteins
DK1871418T3 (en) 2005-04-19 2014-06-10 Seattle Genetics Inc Humanized anti-CD70 binding agents and uses thereof
PL1879901T3 (en) 2005-04-21 2010-06-30 Medimmune Ltd Pyrrolobenzodiazepines
EP1931671B1 (en) 2005-10-05 2009-04-08 Spirogen Limited Alkyl 4- [4- (5-oxo-2, 3, 5, 11a-tetrahyd0-5h-pyrr0l0 [2, 1-c][1, 4]benzodiazepine-8-yloxy) -butyrylamino]-1h-pyrrole-2-carboxylate derivatives and related compounds for the treatment of a proliferative disease
CA3052368A1 (en) 2005-10-07 2007-04-19 Exelixis, Inc. Azetidines as mek inhibitors
PT1813614E (en) 2006-01-25 2012-01-09 Sanofi Sa Cytotoxic agents comprising new tomaymycin derivatives
KR101528939B1 (en) 2006-07-18 2015-06-15 사노피 Antagonist antibody against epha2 for the treatment of cancer
US20080112961A1 (en) 2006-10-09 2008-05-15 Macrogenics, Inc. Identification and Engineering of Antibodies with Variant Fc Regions and Methods of Using Same
EP1914242A1 (en) 2006-10-19 2008-04-23 Sanofi-Aventis Novel anti-CD38 antibodies for the treatment of cancer
PT2099823E (en) 2006-12-01 2014-12-22 Seattle Genetics Inc Variant target binding agents and uses thereof
EP2144628B1 (en) 2007-05-08 2012-10-17 Genentech, Inc. Cysteine engineered anti-muc16 antibodies and antibody drug conjugates
PT2019104E (en) 2007-07-19 2013-12-03 Sanofi Sa Cytotoxic agents comprising new tomaymycin derivatives and their therapeutic use
RU2505544C2 (en) 2007-10-19 2014-01-27 Дженентек, Инк. Antibodies against tenb2 constructed with cysteine, and conjugates, antibody-drug
NO2842575T3 (en) 2008-03-18 2018-02-24
GB0813432D0 (en) 2008-07-22 2008-08-27 Spirogen Ltd Pyrrolobenzodiazepines
CN102176873A (en) 2008-10-15 2011-09-07 捷迈有限公司 Intramedullary nail
GB0819095D0 (en) 2008-10-17 2008-11-26 Spirogen Ltd Pyrrolobenzodiazepines
GB0819097D0 (en) 2008-10-17 2008-11-26 Spirogen Ltd Pyrrolobenzodiazepines
US8426402B2 (en) 2009-02-05 2013-04-23 Immunogen, Inc. Benzodiazepine derivatives
FR2949469A1 (en) 2009-08-25 2011-03-04 Sanofi Aventis ANTICANCER DERIVATIVES, THEIR PREPARATION AND THEIR THERAPEUTIC APPLICATION
SG178602A1 (en) 2009-09-01 2012-04-27 Abbott Lab Dual variable domain immunoglobulins and uses thereof
WO2011038159A2 (en) 2009-09-24 2011-03-31 Seattle Genetics, Inc. Dr5 ligand drug conjugates
WO2011100227A1 (en) 2010-02-09 2011-08-18 Bristol-Myers Squibb Company Benzylpyrrolidinone derivatives as modulators of chemokine receptor activity
IT1398164B1 (en) 2010-02-10 2013-02-14 Givi S R L Unipersonale COUPLING SYSTEM FOR SUITCASES FOR MOTORCYCLES
CN103068405A (en) 2010-04-15 2013-04-24 西雅图基因公司 Targeted pyrrolobenzodiazapine conjugates
CN102971329B (en) 2010-04-15 2016-06-29 麦迪穆有限责任公司 Pyrrolobenzodiazepines for treating proliferative disease is tall and erect
WO2011130615A2 (en) 2010-04-15 2011-10-20 Dr. Reddy's Laboratories Ltd. Preparation of lacosamide
GB201006340D0 (en) 2010-04-15 2010-06-02 Spirogen Ltd Synthesis method and intermediates
EA024730B1 (en) 2010-04-15 2016-10-31 Медимьюн Лимитед Pyrrolobenzodiazepine compounds, conjugates thereof, pharmaceutical compositions comprising said conjugates, and use of said conjugates
WO2012112708A1 (en) 2011-02-15 2012-08-23 Immunogen, Inc. Cytotoxic benzodiazepine derivatives and methods of preparation
AU2012311505B2 (en) 2011-09-20 2016-09-29 Medimmune Limited Pyrrolobenzodiazepines as unsymmetrical dimeric PBD compounds for inclusion in targeted conjugates
CA2850373C (en) 2011-10-14 2019-07-16 Seattle Genetics, Inc. Pyrrolobenzodiazepines and targeted conjugates
KR101877598B1 (en) 2011-10-14 2018-07-11 메디뮨 리미티드 Pyrrolobenzodiazepines and conjugates thereof
MX341523B (en) 2011-10-14 2016-08-24 Medimmune Ltd Pyrrolobenzodiazepines.
EP2751076B1 (en) 2011-10-14 2018-04-25 MedImmune Limited Synthesis method and intermediates useful in the preparation of pyrrolobenzodiazepines
CN110183470A (en) 2011-10-14 2019-08-30 麦迪穆有限责任公司 Pyrrolobenzodiazepines are tall and erect
KR101961976B1 (en) 2011-10-14 2019-03-25 시애틀 지네틱스, 인크. Pyrrolobenzodiazepines and targeted conjugates
SG11201500093TA (en) 2012-07-09 2015-02-27 Genentech Inc Immunoconjugates comprising anti-cd79b antibodies
TW201406785A (en) 2012-07-09 2014-02-16 Genentech Inc Anti-CD22 antibodies and immunoconjugates
EP2879708A4 (en) 2012-08-02 2016-03-16 Genentech Inc Anti-etbr antibodies and immunoconjugates
LT2839860T (en) 2012-10-12 2019-07-10 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
TR201815418T4 (en) 2012-10-12 2018-11-21 Adc Therapeutics Sa Pyrrolobenzodiazepine -anti-psma antibody conjugates.
AU2013328674B2 (en) 2012-10-12 2017-06-22 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9745303B2 (en) 2012-10-12 2017-08-29 Medimmune Limited Synthesis and intermediates of pyrrolobenzodiazepine derivatives for conjugation
EP2935268B2 (en) * 2012-12-21 2021-02-17 MedImmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9821074B2 (en) 2013-03-13 2017-11-21 Genentech, Inc. Pyrrolobenzodiazepines and conjugates thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7514078B2 (en) * 2001-06-01 2009-04-07 Cornell Research Foundation, Inc. Methods of treating prostate cancer with anti-prostate specific membrane antigen antibodies

Also Published As

Publication number Publication date
RS57694B1 (en) 2018-11-30
WO2014057113A1 (en) 2014-04-17
US20150283258A1 (en) 2015-10-08
TR201815418T4 (en) 2018-11-21
BR112015008173A2 (en) 2017-11-28
PL2906253T3 (en) 2019-02-28
CA2887894A1 (en) 2014-04-17
AU2013328619A1 (en) 2015-04-23
EP2906253A1 (en) 2015-08-19
KR101986404B1 (en) 2019-06-07
SI2906253T1 (en) 2018-11-30
JP6270859B2 (en) 2018-01-31
CA2887894C (en) 2019-10-29
CN105102003A (en) 2015-11-25
MX364326B (en) 2019-04-23
NZ707486A (en) 2018-09-28
EP2906253B9 (en) 2019-04-10
DK2906253T3 (en) 2018-10-22
HRP20181646T1 (en) 2018-12-14
MX2015004416A (en) 2015-10-15
AU2013328619B2 (en) 2016-11-17
KR20150085813A (en) 2015-07-24
HUE041274T2 (en) 2019-05-28
ES2690067T3 (en) 2018-11-19
US10751346B2 (en) 2020-08-25
PT2906253T (en) 2018-11-05
LT2906253T (en) 2018-10-10
CY1120916T1 (en) 2019-12-11
ES2690067T9 (en) 2019-06-19
HRP20181646T2 (en) 2019-08-09
EP2906253B1 (en) 2018-08-01
JP2015534577A (en) 2015-12-03

Similar Documents

Publication Publication Date Title
CN105102003B (en) Tall and erect-anti-PSMA antibody conjugates of Pyrrolobenzodiazepines
CN105102004B (en) Pyrrolobenzodiazepines Zhuo-anti-CD22 antibody conjugate
CN105102068B (en) Pyrrolobenzodiazepines Zhuo-antibody conjugates
CN105050661B (en) Pyrrolobenzodiazepines Zhuo antibody conjugates
CN104955485B (en) Pyrrolobenzodiazepines Zhuo Anti-HER 2 conjugate
EP2906250A1 (en) Pyrrolobenzodiazepine-anti-psma antibody conjugates
EP2906297A1 (en) Pyrrolobenzodiazepine-antibody conjugates
CN108093640A (en) Site-specific antibodie-drug conjugate
CN107683146A (en) Site-specific antibodie drug conjugate
CN107548306A (en) Site-specific antibodie drug conjugate

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20190305

Termination date: 20201011